Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS

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DOI: 10.1371/journal.pbio.1000614 · Source: PubMed
Abstract
TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.
Molecular Determinants and Genetic Modifiers of
Aggregation and Toxicity for the ALS Disease Protein
FUS/TLS
Zhihui Sun
1.
, Zamia Diaz
2.
, Xiaodong Fang
1
, Michael P. Hart
1
, Alessandra Chesi
1
, James Shorter
2
*,
Aaron D. Gitler
1
*
1 Department of Cell and Developmental Biology, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 2 Department
of Biochemistry and Biophysics, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America
Abstract
TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis
(ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD.
However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a
yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in
the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule
compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and
filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like
domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In
further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens
uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our
findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via
distinct mechanisms. These differences will likely have important therapeutic implications.
Citation: Sun Z, Diaz Z, Fang X, Hart MP, Chesi A, et al. (2011) Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease
Protein FUS/TLS. PLoS Biol 9(4): e1000614. doi:10.1371/jo urnal.pbio.1000614
Academic Editor: Jonathan S. Weissman, University of California San Francisco/Howard Hughes Medical Institute, United States of America
Received September 16, 2010; Accepted March 17, 2011; Published April 26, 2011
Copyright: ß 2011 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the Packard Center for ALS Research at Johns Hopkins (A.D.G. and J.S.), an NIH Director’s New Innovator
Award 1DP2OD004417-01 (A.D.G), NIH R01 NS065317 (A.D.G.), an NIH Director’s New Innovator Award 1DP2OD002177-01 (J.S.), NIH R21 NS067354-0110 (J.S.), a
University of Pennsylvania Diabetes and Endocrinology Research Center Pilot and Feasibility grant, and an Ellison Medical Foundation New Scholar in Aging
Award (J.S.). A.D.G. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: ALS, amyotr ophic lateral sclerosis; EM, electron microscopy; FTLD, frontotemporal lobar degeneration; FUS, fused in sarcoma; GPD,
glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; NLS, nuclear localization signal; PABP, polyA-binding protein; P-bodies, processing
bodies; PrD, prion-like domain; PrP, prion protein; RRM, RNA recognition motif; SCA, spinocerebellar ataxia; SGA, synthetic genetic array; TDP, TAR-DNA-binding
protein; TEV, tobacco etch virus; Tif, translation initiation factor; TLS, translocated in liposarcoma; WT, wild-type; YFP, yellow fluorescent protein
* E-mail: gitler@mail.med.upenn.edu (ADG); jshorter@mail.med.upenn.edu (JS)
. These authors contributed equally to this work.
Introduction
Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig’s
disease, is a devastating neurodegenerative disease. It is a rapidly
progressing motor neuron wasting disorder that leads to paralysis
and death typically within 2–5 years of onset. There are no cures
or effective treatments. Given the similarities in presentation and
pathology of familial and sporadic disease, study of genes mutated
in familial disease can shed light on mechanisms of both familial
ALS and the more common sporadic form. The first familial gene
associated with ALS was SOD1 [1], and much research over the
past 10–15 years has focused on mechanisms by which mutant
SOD1 may cause motor neuron dysfunction and loss [2].
Insight into ALS changed dramatically in 2006 when the 43 kDa
TAR-DNA-binding protein (TDP-43) was identified as a protein
that accumulates abnormally in the ubiquitinated pathological
lesions that characterize brain and spinal cord tissue of almost every
non-SOD1 ALS patient [3–5]. Similar TDP-43 inclusions were also
identified in degenerating neurons in a subset of frontotemporal
lobar degeneration (FTLD-TDP) cases [3–5]. TDP-43 is an RNA-
binding protein with two RNA recognition motifs (RRMs) and a
glycine rich domain [6]. In 2008, several groups independently
reported the identification of over 30 different mutations in the
TDP-43 gene (TARDBP) in various sporadic and familial ALS
patients [6–10]. TDP-43 mutations were subsequently identified in
various FTLD-TDP cases [11,12]. Taken together, these studies
strongly suggest that TDP-43 is a new human neurodegenerative
disease protein. Wild-type (WT) TDP-43 accumulates abnormally
in cytoplasmic, ubiquitinated inclusions in degenerating neurons of
ALS and FTLD-TDP patients, and mutations in the TDP-43 gene
are linked with disease in rare familial and sporadic cases. Despite
these advances, how TDP-43 contributes to disease, which domain
of TDP-43 drives aggregation, and how ALS-linked mutations
affect TDP-43 function and aggregation remained unclear.
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To address these deficits, we investigated the pathogenic
properties of TDP-43 in yeast. The yeast system is simple and
fast and has highly conserved fundamental pathways that allow
powerful insights into complex human neurodegenerative diseases
such as Parkinson’s disease, Alzheimer’s disease, and ALS [13].
Therefore, we developed a yeast model of TDP-43 to study TDP-
43 biology as well as the mechanisms of TDP-43 aggregation and
toxicity. Expression of human TDP-43 in yeast resulted in
cytoplasmic aggregation and toxicity, thus modeling key aspects
of human TDP-43 proteinopathies. These studies revealed that
RRM2 and the C-terminal domain of TDP-43 (Figure 1A) are
required for aggregation and toxicity [14]. Notably, all but one of
over 30 ALS-linked mutations reside in the C-terminal domain,
which the yeast system defined as critical for toxicity. Moreover, a
combination of pure protein studies and in vivo analyses in yeast
demonstrated that ALS-linked TDP-43 mutations render TDP-43
more aggregation-prone and enhance toxicity [15]. These studies
demonstrated that the aggregation propensity and severity of
toxicity of TDP-43 variants observed in ALS could be recapitu-
lated in yeast. Moreover, we have discovered a potent genetic
modifier of TDP-43 toxicity in yeast, Pbp1, which is connected
with ALS in humans [16]. The human homolog of Pbp1, ataxin 2,
harbors a polyglutamine tract that is greatly expanded (.34
glutamines) in spinocerebellar ataxia type 2 [16]. Importantly,
intermediate-length polyQ expansions (,27–33 glutamines) in
ataxin 2 are a significant genetic risk factor for ALS in humans
[16]. Clearly, the power of yeast genetics can be exploited to
define basic disease mechanisms of fundamental importance to
human neurodegenerative disease.
Shortly following the identification of mutations in TDP-43 in
ALS, mutations in another gene encoding an RNA-binding
protein, FUS (fused in sarcoma; also known as TLS, translocated
in liposarcoma), were connected to familial ALS [17,18].
Additional mutations in FUS have recently been identified in
sporadic ALS cases and in a subset of frontotemporal lobar
degeneration (FTLD-FUS) cases [19,20]. FUS is normally a
nuclear protein, but ALS patients harboring FUS mutations
exhibit prominent neuronal cytoplasmic FUS accumulations that
appear devoid of TDP-43 [18]. Several other examples of
neurodegenerative disease are beginning to emerge where the
predominant disease phenotype is the cytoplasmic aggregation of
wild-type FUS. These include some cases of juvenile ALS [21],
basophilic inclusion body disease [22], as well as the majority of
tau- and TDP-43-negative frontotemporal lobar degeneration
cases [23]. Moreover, FUS is also aggregated in Huntington’s
disease; spinocerebellar ataxia (SCA) type 1, 2, and 3; and
dentatorubropallidoluysian atrophy [24,25]. These findings extend
the spectrum of disorders associated with FUS aggregation beyond
ALS and FTLD-FUS and suggest the importance of understand-
ing mechanisms of aggregation of WT as well as mutant FUS.
FUS was initially discovered as part of a chromosomal
translocation associated with human myxoid liposarcomas [26].
Subsequent studies have revealed roles for FUS in transcription,
RNA processing, and RNA transport [27–29]. In neurons, FUS is
localized to the nucleus but is transported to dendritic spines at
excitatory post-synapses in a complex with RNA and other RNA-
binding proteins [30]. In further support of a role of FUS in
maintaining neuronal architecture, primary hippocampal neurons
cultured from FUS knockout mouse embryos display defects in
spine morphology and decreased spine density [31]. It remains
unclear, however, how loss of this function of FUS or perhaps a
novel toxic gain-of-function associated with FUS mutations
contribute to ALS. Importantly, it is also uncertain whether
FUS is intrinsically aggregation-prone. Indeed, FUS might simply
be a marker of disease that is sequestered by other aggregated
components.
FUS and TDP-43 possess a similar domain structure. Like
TDP-43, FUS has an RRM and a glycine-rich domain (Figure 1A).
Moreover, using a bioinformatic algorithm designed to identify
yeast prion domains [32], we recently identified novel ‘‘prion-like’’
domains in the N-terminal domain of FUS (amino acids 1–239)
and in the C-terminal domain of TDP-43 (amino acids 277–414)
(Figure 1A, Figure S1) [33]. Similar to prion domains found in
yeast prion proteins such as Sup35, Ure2, and Rnq1, this domain
is enriched in uncharged polar amino acids (especially asparagine,
glutamine, and tyrosine) and glycine [32,34]. This type of domain
encodes all the information necessary to form a prion in yeast [32–
34]. It should be noted, however, that this type of domain is not
found in all prion proteins, including HET-s from Podospora anserina
and mammalian prion protein (PrP) [33,34]. Remarkably, by
using this bioinformatic algorithm [32] to score and rank the
human proteome (27,879 human proteins) for prion-like proper-
ties, FUS and TDP-43 ranked 15
th
and 69
th
, respectively [33].
Our findings raise the intriguing possibility that RRM proteins
with predicted prion-like domains may be particularly relevant to
ALS [15,33,35,36]. Virtually all the ALS-linked mutations in
TDP-43 lie in its prion-like domain [33]. By contrast, only a few of
the ALS-linked mutations in FUS lie in its prion-like domain [33].
Indeed, the majority of ALS-linked FUS mutations reside at the
extreme C-terminal region [37]. The identification of two RNA-
binding proteins with a similar domain architecture that aggregate
and are sometimes mutated in ALS and FTLD gives rise to the
emerging concept that RNA metabolic pathways may play a
major role in ALS and FTLD pathogenesis [38].
Despite these similarities between TDP-43 and FUS, it is
unknown whether TDP-43 and FUS aggregate and cause toxicity
by similar mechanisms. Here, we address this issue and establish,
for the first time, two vital weapons in the fight against FUS
proteinopathies, which have been critical in advancing our basic
understanding of various other protein misfolding disorders,
including Parkinson’s disease, Huntington’s disease, and TDP-43
proteinopathies [13–16,39–45]. First, we establish a simple yeast
model of FUS aggregation and toxicity. Second, we reconstitute
Author Summary
Many human neurodegenerative diseases are associated
with the abnormal accumulation of protein aggregates in
the neurons of affected individuals. Amyotrophic lateral
sclerosis (ALS), also known as Lou Gehrig’s disease, is a
fatal human neurodegenerative disease caused primarily
by a loss of motor neurons. Recently, mutations in a gene
called fused in sarcoma (FUS) were identified in some ALS
patients. The basic mechanisms by which FUS contributes
to ALS are unknown. We have addressed this question
using protein biochemistry and the genetically tractable
yeast Saccharomyces cerevisiae. We defined the regions of
biochemically pure FUS protein that contribute to its
aggregation and toxic properties. We then used genome-
wide screens in yeast to identify genes and cellular
pathways involved in the toxicity of FUS. Many of the
FUS toxicity modifier genes that we identified in yeast
have clear homologs in humans, suggesting that these
might also be relevant for the human disease. Together,
our studies provide novel insight into the basic mecha-
nisms associated with FUS aggregation and toxicity.
Moreover, our findings open new avenues that could be
explored for therapeutic intervention.
Mechanisms of FUS Aggregation and Toxicity
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Figure 1. Yeast FUS model. (A) Schematic of domain architecture of FUS and TDP-43. Note that both contain glycine-rich regions, RRM, and prion-
like domains. In addition, FUS has two RGG domains. (B) Schematic of galactose-inducible construct to express human FUS fused to YFP. (C) Yeast
cells expressing YFP alone or YFP-tagged human RNA-binding proteins. Not all RNA-binding proteins aggregate when expressed in yeast. For
example, three related human RRM-containing proteins did not form inclusions when expressed in yeast. Instead they were diffusely localized: PPIE
localized to the nucleus and cytoplasm; DND1 localized to the cytoplasm; and DNAJC17 was restricted to the nucleus. The ALS disease proteins, TDP-
43 and FUS, formed multiple cytoplasmic foci when expressed in yeast. (D) Immunoblot showing untagged and YFP-tagged FUS expression. (E) FUS is
toxic when expressed in yeast cells compared to YFP alone control. 5-fold serial dilutions of yeast cells expressing YFP alone, untagged FUS, or YFP-
tagged FUS. Because of the galactose-inducible promoter, FUS expression is repressed when cells are grown in the presence of glucose (left panel,
FUS expression ‘‘off’’) and induced when grown in the presence of galactose (right panel, FUS expression ‘‘on’’). (F) Fusing the strong heterologous
SV40 NLS to the N-terminus of FUS restricts it mostly to the nucleus. (G) Spotting assay shows that nuclear-localized SV40 FUS-YFP is less toxic than
WT FUS.
doi:10.1371/journal.pbio.1000614.g001
Mechanisms of FUS Aggregation and Toxicity
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FUS misfolding and aggregation using pure protein. These two
approaches have served as important foundations for understand-
ing mechanistic aspects of numerous neurodegenerative disorders
and have empowered countless advances. We establish that, as for
TDP-43, the RRM and the prion-like domain of FUS are required
for aggregation and toxicity in yeast. However, in contrast to
TDP-43, we find that additional determinants within the first
RGG domain (Figure 1A) are also critical for FUS aggregation
and toxicity. Importantly, we demonstrate that pure FUS is
inherently aggregation-prone in the absence of other components
and this behavior requires determinants in the prion-like domain
and first RGG domain of FUS (Figure 1A). Aggregates formed by
pure FUS are filamentous and resemble those formed by FUS in
degenerating motor neurons of ALS patients. ALS-linked TDP-43
mutations can promote aggregation in vitro with pure proteins and
in yeast [15]. By contrast, we find that ALS-linked FUS mutations
do not promote aggregation per se. Finally, using two genome-
wide screens in yeast, we identified several genes and pathways as
potent modifiers of FUS toxicity. Many of the genes that we
discovered in the yeast screens have human homologs. Thus, they
are likely to provide insight into the specific cellular pathways
perturbed by FUS accumulation and may ultimately suggest novel
avenues for therapeutic investigation. Surprisingly, almost all of
the genetic modifiers had no effect on TDP-43 toxicity in yeast.
These key differences between FUS and TDP-43 will help guide
the design of therapeutic interventions aimed at mitigating FUS
aggregation in disease.
Results
FUS Forms Inclusions in the Yeast Cytoplasm and Is Toxic
To model aspects of FUS pathology in yeast, we first
transformed yeast cells with a high-copy 2 micron (2
m) plasmid
containing human FUS fused to the yellow fluorescent protein
(YFP; Figure 1B). Because TDP-43 was toxic to yeast [14], we
placed FUS-YFP expression under the control of a tightly
regulated galactose-inducible promoter (Figure 1B) to prevent
deleterious effects during routine passage. After growing transfor-
mants in non-inducing conditions (raffinose media), we induced
expression of FUS-YFP in galactose-containing media. Overex-
pression is a common tool to study the aggregation and toxicity of
numerous proteins ranging from alpha-synuclein to TDP-43
[14,39,45]. It provides a method to elicit protein misfolding by
increasing protein concentration and exceeding proteostatic
buffers [46]. Moreover, overexpression is likely to yield key
information because an established cause of several human
neurodegenerative diseases is increased expression of aggrega-
tion-prone proteins, such as alpha-synuclein, amyloid precursor
protein, and TDP-43 [47–49]. Following 4–6 h of induction, we
visualized FUS-YFP localization by fluorescence microscopy
(Figure 1C). Whereas the control, YFP alone, was localized
diffusely throughout the cytoplasm and nucleus, FUS-YFP
localized to the cytoplasm where it formed numerous foci
(Figure 1C). FUS-YFP showed a similar cytoplasmic localization
pattern when expressed from a low-copy galactose-inducible CEN
plasmid (unpublished data). The FUS localization pattern was
strikingly similar to that of TDP-43 in yeast (Figure 1C and [14]),
in terms of size, shape, and quantity of foci in the cytoplasm
(Figure 1C). Indeed when co-expressed in the same cell, FUS-YFP
and TDP-43-CFP co-localized to the same cytoplasmic foci
(Figure S2). Thus, TDP-43 and FUS inclusions partition to a
similar compartment in yeast.
Next, we employed a weaker promoter (glyceraldehyde-3-
phosphate dehydrogenase (GPD) promoter) to express FUS at
lower levels. Here, FUS-YFP localized to both the nucleus and
cytoplasm, where it was diffusely distributed (Figure S3). Similar
results were seen with even weaker yeast promoters (CYC1 and
NOP1; unpublished data). Thus, the FUS expression level plays a
key role in FUS localization and aggregation in yeast. These data
predict that sequence variants or copy number variants in the FUS
gene that increase FUS expression might also contribute to ALS,
FTLD-FUS, and other FUS proteinopathies. Indeed, a variant in
the 39UTR of the TDP-43 gene increases TDP-43 expression and
contributes to FTLD-TDP [47]. Moreover, motor neurons express
higher levels of FUS than other tissues, which might render them
more vulnerable to FUS misfolding events [50].
In mammalian cells, FUS is normally restricted to the nucleus
[51–55]. By contrast, in yeast, FUS is mostly localized to the
cytoplasm. This difference suggests that the non-canonical FUS
nuclear localization signal (NLS; amino acids 500–526) might not
be very efficient in yeast. Indeed, in an accompanying manuscript,
Ju et al. present data that directly support this hypothesis [56].
Alternatively, FUS might require post-translational modifications
to localize to the nucleus, which do not occur in yeast. In an effort
to restrict FUS to the nucleus, we fused a strong heterologous NLS
(the SV40 NLS [57]) to the N-terminus of FUS. The SV40 NLS
was sufficient to largely restrict FUS to the nucleus, but some
cytoplasmic localization was also observed (Figure 1F). Impor-
tantly, restricting FUS to the nucleus eliminated aggregation
(Figure 1F). Thus, FUS accumulation in the cytoplasm contributes
to its aggregation. Despite the differences between FUS localiza-
tion in yeast and mammalian cells, we can clearly use the
genetically tractable yeast system to model FUS cytoplasmic
aggregation, a critical pathological event in ALS and FTLD
[17,18]. Furthermore, defective nuclear import of FUS might be a
key upstream event in ALS [52].
Having established that FUS, like TDP-43, forms cytoplasmic
inclusions when expressed in yeast, we next asked if cytoplasmic
aggregation of FUS was toxic. To assess FUS toxicity, we performed
spotting assays on galactose media. Expressing FUS-YFP or
untagged FUS inhibited growth, whereas YFP had no effect
(Figure 1E). Thus, as for TDP-43, FUS expression in yeast was
cytotoxic. Cytotoxicity correlated positively with cytoplasmic
aggregation. First, expressing FUS at lower levels from the GPD
promoter did not induce cytoplasmic FUS inclusions (Figure S3) and
did not confer toxicity (unpublished data). Second, restricting FUS to
the nucleus with the SV40 NLS (Figure 1F) greatly reduced toxicity
(Figure 1G). These data suggest that cytoplasmic FUS aggregation is
a critical pathological event in ALS and that neurodegeneration
might be caused by a toxic gain of function in the cytoplasm.
Importantly, not every human RNA-binding protein aggregates
and is toxic when expressed at high levels in yeast. Indeed, we
expressed 132 human proteins containing RRMs in yeast. Of these,
35 (including TDP-43 and FUS) aggregated and were toxic (A.D.G.
unpublished observations; Figure 1C). It will be important to
determine whether any of these RRM-bearing proteins, aside from
FUS and TDP-43, are connected to neurodegenerative disease.
Moreover, it will be important to define whether common sequence
determinants among these 35 RRM-bearing proteins promote
aggregation and toxicity. One striking feature of FUS and TDP-43,
as well as at least seven other human RNA-binding proteins that are
toxic and aggregate in yeast, is the presence of a prion-like domain
(Figure 1A; A.D.G. unpublished observations) [33].
FUS Associates with Stress Granules and P-Bodies in
Yeast
We noticed that FUS-YFP cytoplasmic accumulations in yeast
are highly dynamic under various growth conditions (Z.S., X.D.F.,
Mechanisms of FUS Aggregation and Toxicity
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and A.D.G., unpublished observations). This dynamic behavior
was reminiscent of RNA processing bodies (P-bodies) and stress
granules. P-bodies and stress granules play important roles in
regulating the translation, degradation, and localization of
mRNAs. The pathways regulating the incorporation of RNAs
and RNA-binding proteins into these structures are highly
conserved from yeast to human [58]. Under various stress
situations, including heat shock and oxidative stress, TDP-43
and FUS localize to these transient subcellular compartments and
sites of RNA processing [59–62]. Moreover, even under normal
conditions some ALS-linked FUS mutants localize to stress
granules [51–55]. Thus, we tested whether FUS could induce
stress granule or P-body formation in yeast and whether FUS
localized to these structures. We expressed FUS-YFP or YFP alone
in yeast cells harboring RFP- or CFP-tagged stress granule or P-
body markers (Figure 2). To detect stress granules we used Pbp1-
CFP and to detect P-bodies we used Dcp2-RFP [63]. Expressing
YFP alone did not affect the localization of the P-body or stress
granule components, which were diffuse under normal conditions
(Figure 2A,B; unpublished data). However, FUS expression
induced the formation of P-bodies and stress granules and FUS-
YFP colocalized with both of these structures (Figure 2A,B). Thus,
Figure 2. FUS associates with stress granules and P-bodies in yeast. (A) Yeast cells expressing YFP alone (top row) or FUS-YFP (bottom row).
Dcp2-RFP was used to monitor P-body formation and localization. FUS-YFP expression induced the formation of P-bodies and FUS-YFP cytoplasmic
localized to these structures. (B) FUS also induced the formation of and localized to stress granules, as monitored by a CFP-fusion to the stress granule
protein Pbp1. Similar results were observed with independent P-body and stress granule markers, Lsm1 and Pub1, respectively (unpublished data).
doi:10.1371/journal.pbio.1000614.g002
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FUS localizes to and induces the formation of RNA granules in
yeast as it does in human cells [51,53–55]. These RNA granule
assembly pathways are highly conserved from human to yeast.
Thus, yeast provides a powerful system to dissect how FUS
associates with these structures and to identify genetic and
chemical modifiers of this process.
Defining the Regions of FUS That Contribute to
Aggregation and Toxicity in Yeast
To determine sequence features of FUS that were sufficient and
necessary for aggregation and toxicity in yeast, we next performed
a structure-function analysis. We recently used a similar approach
for TDP-43 and determined that the C-terminal prion-like domain
was required for aggregation and toxicity [14]. Underscoring the
power of this approach, similar results have been reported for the
C-terminal domain of TDP-43 in mammalian cells and in animal
models [64,65]. Moreover, all but one of the recently identified
human ALS-linked TDP-43 mutations are located in this same C-
terminal region [6]. We generated a series of FUS truncations
(Figure 3A). We expressed each of the truncated FUS constructs as
YFP-fusions and determined their subcellular localization
(Figure 3B) and toxicity (Figure 3D). Immunoblotting confirmed
that all of the fusion proteins were expressed at comparable levels
(Figure 3C; unpublished data).
Full-length FUS formed multiple cytoplasmic inclusions in yeast
(Figures 1C, 3B). Interestingly, removing the last 25 residues of
FUS, which harbor most of the ALS-linked mutations [37], did
not affect aggregation (Figure 3B, construct 1–501). This result is
consistent with a recent report that a similar FUS truncation
mutant (R495X) is connected with a severe ALS phenotype [51].
A larger C-terminal deletion also had little effect on cytoplasmic
aggregation (Figure 3B, construct 1–453). Thus, C-terminal
portions of FUS are not essential for cytoplasmic aggregation.
For TDP-43, the C-terminal prion-like domain is necessary but
not sufficient for cytoplasmic aggregation [14]. TDP-43 also requires
a portion of RRM2 (Figure 1A) [14]. However, for FUS, the N-
terminal prion-like domain and the RRM resulted in an entirely
nuclear localized protein (Figure 3B, construct 1–373; Figure S4).
Adding back the first RGG domain (amino acids 371–422) was
sufficient to restore cytoplasmic aggregation (Figure 3B, construct 1–
422). Thus, in contrast to our findings with TDP-43 [14], the prion-
like domain and the RRM of FUS (Figure 3B, construct 1–373) were
insufficient to confer cytoplasmic aggregation. Additional C-terminal
determinants within the first RGG domain are required to confer
cytoplasmic aggregation (Figure 3B, construct 1–422).
Next, we asked if deletion of portions of the N-terminal prion-
like domain of FUS, which spans the QGSY-rich domain and a
portion of the Gly-rich domain (amino acids 1–239) (Figure 1A),
prevented aggregation. Indeed, the generation of large cytoplasmic
inclusions required most of the N-terminal QGSY-rich domain
(Figure 3B, compare constructs 1–501, 50–526, 100–526, and
165–526) (Figure 3A,B). Deletion of the entire N-terminal QGSY-
rich domain (construct 165–526) yielded mostly diffuse cytoplas-
mic staining with occasional small foci (Figure 3A,B). However,
shorter N-terminal constructs comprising just the N-terminal
QGSY-rich domain or this domain plus the Gly-rich domain did
not aggregate and were localized in the nucleus (Figure 3B,
constructs 1–168 and 1–269; Figure S4). Thus, the N-terminal
prion-like domain of FUS is necessary but not sufficient for
aggregation. Rather, FUS requires sequences in both the N-
terminal region and the C-terminal region for robust formation of
large cytoplasmic inclusions. Accordingly, large N-terminal
deletions were diffusely localized within the cytoplasm, with only
occasional small cytoplasmic puncta (Figure 3B, constructs 165–
526, 267–526, 285–526, and 368–526). Thus, in distinction to
TDP-43, which requires its C-terminal prion-like domain and a
portion of RRM2 (Figure 1A) to aggregate in yeast [14], FUS
requires its N-terminal prion-like domain, RRM, and first RGG
domain to aggregate in yeast (Figures 1A, 3A). This key difference
will have important implications for the design of therapeutic
strategies aimed at preventing or reversing aggregation.
The Doma ins of FUS Required for Aggregation in Yeast
Contribute to Aggregation in Mammalian Cells
Our domain mapping experiments in yeast indicate that the first
RGG domain of FUS (amino acids 371–422) is important for
driving aggregation (e.g., Figure 3B, compare constructs 1–373
and 1–422) and that sequences in the N-terminal prion-like
domain (amino acids 1–239) are also important (e.g., Figure 3B,
compare constructs 50–526 and 165–526). To test these
predictions in mammalian cells, we transfected several of these
deletion constructs (as C-terminal V5 epitope tag fusions) in COS-
7 cells. In contrast to yeast cells, where full-length FUS (construct
1–526) forms cytoplasmic inclusions, and consistent with previous
reports in mammalian cells [17,18,51,52], full-length FUS
localized almost exclusively to the nucleus, forming occasional
cytoplasmic foci (Figure 4). This difference between the localiza-
tion of full-length FUS in yeast (almost entirely cytoplasmic and
forms inclusions) versus mammalian cells (almost entirely nuclear
and diffuse) is also seen with TDP-43 (e.g., compare [66] and [14])
and might reflect differences in the efficacy of the FUS and TDP-
43 nuclear localization signals in yeast and mammals. Indeed, Ju et
al. demonstrate that the FUS NLS (amino acids 500–526) is
ineffective in yeast [56].
Consistent with our yeast data, FUS constructs 1–269 and 1–
373 localized almost exclusively to the nucleus in a diffuse pattern,
although there was more cytoplasmic staining with 1–373
(Figure 4). These results were surprising since these constructs
lack the C-terminal NLS defined in other studies [52,54].
However, these results are consistent with those of Kino et al.,
who find that FUS 1–278 is localized to the nucleus and FUS 1–
360 is localized to the nucleus as well as the cytoplasm [54]. These
data suggest that additional determinants of nuclear localization
exist in the FUS primary sequence. Indeed, scanning the FUS
primary sequence using NLStradumus [67] revealed three NLS
sequences in FUS comprising residues 241–251, 381–395, and
480–521. These two additional NLS sequences (241–251 and
381–395) might help explain why all of the FUS constructs in
Figure 4 have some ability to localize to the nucleus.
Strikingly, as we observed in yeast, addition of the first RGG
domain (construct 1–422) resulted in prominent cytoplasmic FUS
aggregation in COS-7 cells (Figure 4). FUS construct 50–526
aggregated in yeast (Figure 3B) and mammalian cells (Figure 4).
However, the morphology of the 50–526 inclusions was distinct
from those formed by 1–422 (one or two large tight inclusions per
cell with 1–422 versus numerous amorphous inclusions with 50–
526). These data indicate that the domains of FUS required for
aggregation in yeast (especially the first RGG domain) are also
critical for FUS aggregation in mammalian cells. Moreover, these
data validate the yeast system as a useful platform for interrogating
mechanisms and genetic modifiers (see below) of FUS aggregation
and toxicity.
Defining the Domains of FUS Required for Toxicity in
Yeast
Having determined the regions of FUS required for aggregation
in yeast, we next determined which regions of FUS contributed to
Mechanisms of FUS Aggregation and Toxicity
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Figure 3. Defining the sequence features contributing to FUS aggregation and toxicity in yeast. (A) A diagram illustrating the domain
structure of FUS along with truncation constructs used in this study. (B) Testing the effects of truncations on FUS localization by fluorescence
microscopy. The C-terminal domain is required for cytoplasmic localization and aggregation (compare constructs 1–373 and 1–526). Arrows point to
larger cytoplasmic FUS inclusions and arrowheads point to cells with more diffuse cytoplasmic FUS with small foci (see table in panel A). (C)
Mechanisms of FUS Aggregation and Toxicity
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toxicity (Figure 3D). As with FUS aggregation, the last 25 amino
acids of FUS, where many of the ALS-linked mutations occur
[37], were not required for toxicity (Figure 3D, construct 1–501).
Indeed, 1–501 was slightly more toxic than full-length FUS
(Figure 3D). This finding is consistent with the severe ALS
phenotype linked to FUS R495X [51]. Similar to TDP-43, the
prion-like domain of FUS was required but not sufficient for
toxicity (Figure 3D, compare constructs 1–526, 1–168, 1–269, and
267–526). As for aggregation, most of the N-terminal prion-like
domain of FUS (amino acids 1–239) was needed for toxicity
(Figure 3D, compare constructs 1–501, 50–526, and 100–526) and
larger N-terminal deletions were not toxic (Figure 3D, compare
Immunoblot showing expression levels of full-length FUS and each truncation. (D) The effects of truncations on toxicity were assessed by spotting
assays. As for aggregation, the C-terminal region is required for toxicity but by itself is not sufficient (construct 368–526). The RRM, glycine-rich region
and most of the prion-like domain (see [33]) are also required for FUS toxicity (compare constructs 1–501, 50–526, and 100–526). RRM, RNA
recognition motif. (E) Mutating conserved phenylalanine residues in the FUS RRM to leucine to abolish RNA binding (FUS
RRM mutant
) does not affect
FUS aggregation in yeast, however RNA binding is important for FUS toxicity because the FUS
RRM mutant
eliminates toxicity in yeast (F).
doi:10.1371/journal.pbio.1000614.g003
Figure 4. FUS domains that contribute to aggregation in mammalian cells. V5-tagged FUS expression constructs were transfected into COS-
7 cells and their localization determined by fluorescence microscopy. Full-length FUS (1–526) localized to the nucleus, consistent with previous
reports [17,18,51,52]. Deletion constructs 1–269 and 1–373 also localized to the nucleus, consistent with our results in yeast (see Figure 2). Also asin
yeast, the addition of sequences in the first FUS RGG domain resulted in FUS aggregation in the cytoplasm (construct 1–422, arrows). Construct 50–
526 also aggregated in the cytoplasm, however the morphology of the inclusions (arrowheads) was distinct from that of 1–422.
doi:10.1371/journal.pbio.1000614.g004
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constructs 165–526, 267–526, 285–526, and 368–526). However,
unlike TDP-43 [14], adding back the RRM to the prion-like
domain did not restore toxicity (Figure 3D, construct 1–373).
Rather, for toxicity the RRM and the first RGG domain were
required in addition to the prion-like domain (Figure 3D, compare
constructs 1–373 and 1–422). However, 1–422 was not as toxic as
full-length FUS, and additional C-terminal sequences were
required to confer full toxicity (Figure 3D, compare constructs
1–422, 1–453, and 1–501). These findings are consistent with a
pathogenic FUS truncation mutant (amino acids 1–466) connected
with sporadic ALS [68].
Next, we tested whether FUS must bind RNA and aggregate to
be toxic in yeast. Thus, we mutated conserved phenylalanine
residues within the FUS RRM to leucine (Phe305, 341, 359,
368Leu) that would disrupt RNA binding [69]. These mutations
were sufficient to mitigate toxicity but had no effect on cytoplasmic
aggregation (Figure 3E,F). Analogous mutations to the RRMs of
TDP-43 disable RNA binding [69] and also mitigate toxicity in
yeast [16]. Taken together, these data indicate that the N-terminal
prion-like domain, first RGG domain, and RRM (likely via RNA
binding) of FUS contribute to toxicity. Identifying the specific
RNA targets of FUS (for example, see [70]) will provide key
insights into mechanisms of toxicity associated with FUS
aggregation in disease. Overall, compared to TDP-43, FUS
aggregation and toxicity in yeast is a more complex multi-domain
process. Importantly, our studies define the prion-like FUS N-
terminal domain and first RGG domain as potential targets to
prevent or reverse FUS aggregation and toxicity.
FUS Is Intrinsically Aggregation Prone
To determine whether FUS is intrinsically prone to aggregation,
we purified bacterially expressed recombinant FUS as a soluble
protein under native conditions. However, expression of various
constructs including N- and C-terminal His-tagged FUS in various
bacterial strains failed to yield soluble protein. The solubility of
various proteins, including TDP-43 and polyglutamine, can be
enhanced by the addition of a glutathione-S-transferase (GST) tag
[15,41,71]. Even so, FUS bearing a C-terminal GST-tag was also
insoluble in various bacterial strains. Fortunately, an N-terminal
GST-tag allowed FUS to be purified as a soluble protein under
native conditions. GST-FUS remained soluble for extended
periods and was competent to bind RNA in mobility shift assays
(Figure 5A). To study FUS aggregation, we added tobacco etch
virus (TEV) protease to cleave at a single unique site and
specifically remove the N-terminal GST-tag (Figure 5B). This
strategy has been utilized successfully to study the aggregation of
extremely aggregation-prone proteins, including polyglutamine
[41,43]. Upon addition of TEV protease, FUS aggregated
extremely rapidly (Figure 5C). By contrast, GST-FUS remained
predominantly soluble (Figure 5C). Under identical conditions
neither GST nor TEV protease aggregated (Figure 5C). Aggre-
gation was dependent on FUS concentration in three ways: at
higher FUS concentrations, the maximum amplitude or endpoint
of turbidity was increased, the length of lag phase was reduced and
the rate of aggregation during assembly phase was accelerated
(Figure 5C). Sedimentation analysis revealed that after addition of
TEV protease, FUS entered the pellet fraction, whereas GST-FUS
remained largely soluble (Figure 5D). Indeed, there was very little
FUS in the supernatant fraction at any time, indicating that
aggregation occurred rapidly after proteolytic liberation of FUS
from GST (Figure 5D). The aggregates formed by FUS did not
react with the amyloid-diagnostic dye Thioflavin-T and were
SDS-soluble, in contrast to those formed by NM, the prion
domain of yeast prion protein Sup35 (Figure 5E,F). Thus, pure
FUS forms aggregates that are likely non-amyloid in nature, just
like the aggregated species of FUS observed in ALS and FTLD
patients [5,72,73].
The rapid aggregation of FUS occurred without agitation of the
reaction (Figures 5C, 6A). Remarkably, under these conditions,
even TDP-43 did not aggregate (Figure 6A). TDP-43 requires
many hours to aggregate unless the reaction is agitated [15].
Agitation had little effect on the rate of FUS aggregation
(Figure 6A,B), indicating that under these conditions FUS
aggregation is energetically favorable. Even when the reaction
was agitated, TDP-43 aggregation was still considerably slower
than FUS aggregation (Figure 6B). In particular, the lag period
prior to aggregation was longer for TDP-43 than for FUS
(Figure 6B). This extended lag period was not due to different rates
of FUS or TDP-43 cleavage by TEV protease, which were
extremely similar (unpublished data). Rather, nucleation of
aggregation is apparently more rate limiting for TDP-43 than it
is for FUS. Collectively, these data suggest that, even in
comparison to TDP-43, FUS is extremely aggregation prone.
These data are also in keeping with the higher prion-like domain
score of FUS compared to TDP-43 [33]. In vivo, such rapid FUS
aggregation is most likely precluded by the proteostasis network
[46]. However, FUS likely escapes these safeguards in disease
situations where proteostatic buffers may have declined with age
or because of environmental triggers. Irrespective of the factors
that may elicit FUS aggregation in disease, pure protein assays
akin to the one we report here have been powerful tools to dissect
the mechanisms underlying the aggregation of various disease-
connected proteins, including TDP-43 and polyglutamine
[15,41,43].
The Prion-Like Domain and First RGG Domain of FUS Are
Important for Aggregation
Next, we determined how the N- and C-terminal domains of
FUS contribute to aggregation of the pure protein. Consistent with
observations in yeast (Figure 3B), deletion of the N-terminal prion-
like domain of FUS yielded protein (267–526) that remained
soluble over the time course of the assay as determined by
turbidity and sedimentation analysis (Figure 7A,B). These data
suggest that the prion-like domain of FUS is required for
aggregation. Curiously, however, but also consistent with obser-
vations in yeast, a protein bearing the prion-like domain and
adjacent C-terminal sequences (1–373) did not aggregate under
these conditions (Figure 7A,B). Even at higher concentrations
(20
mM), neither FUS 267–526 nor FUS 1–373 aggregated.
Moreover, if the reaction was subsequently agitated at 700 rpm for
an additional 60 min neither FUS 267–526 nor FUS 1–373
aggregated.
Next, we tested FUS 1–422, a minimal fragment of FUS able
to confer toxicity and aggregation in yeast (Figure 3B,D). FUS
1–422 aggregated with similar kinetics to full-length FUS as
determined by sedimentation analysis (Figure 7B). Curiously,
however, at these concentrations (2.5–5
mM) FUS 1–422
aggregates did yield a signal by turbidity (Figure 7A). Higher
concentrations of FUS 1–422 (20
mM) were required to
generate aggregates detectable by turbidity (Figure 7A). These
concentration differences in the turbidity measurements for full-
length FUS and FUS 1–422 su ggest that there are large
disparities in the sizes of the aggregates formed by these two
proteins because turbidity readily d etects large but not small
aggregates [74–76]. A similar finding has been made with PrP,
where d eletion of the N-terminal domain reduces the formation
of larger turbid aggregates, without affecting the formation of
smaller aggregates [74]. These data suggest that the C-terminal
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region, comprising amino acids 423–526, while dispensable for
aggregation per se (Figure 7B), promotes the formation of large
macroscopic aggregates of FUS that are detected by turbidity
(Figure 7A).
Pure FUS Aggrega tes Resembl e FUS Aggregates in
Degenerating Neurons of ALS Patients
Electron microscopy (EM) confirmed that pure FUS 1–373 and
FUS 267–526 do not form aggregated species in isolation
Figure 5. FUS is intrinsically aggregation prone. (A) RNA mobility shift experiments.
32
P-labelled FUS RNA probe (see Materials and Methods)
was incubated in the presence or absence of increasing amounts of GST-FUS or GST and resolved on a native gel to observe free and bound RNA
species. (B) Schematic of FUS aggregation assay. TEV protease is added to remove the GST tag and untagged FUS aggregation kinetics are followed
over 90 min. (C) GST-FUS or GST (2.5–5
mM) was incubated in the presence or absence of TEV protease at 22uC for 0–90 min. Turbidity measurements
were taken every minute to assess the extent of aggregation. Values represent means (n = 3). (D) GST-FUS (5
mM) was incubated in the presence or
absence of TEV protease at 22uC for 0–60 min. At the indicated times, reactions were processed for sedimentation analysis. Pellet and supernatant
fractions were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. A representative gel is shown. Note that cleaved FUS partitions
mostly to the pellet fraction, whereas GST-FUS remains in the supernatant (SN) fraction. The amount of GST-FUS or FUS in the pellet fraction was
determined by densitometry in comparison to known quantities of GST-FUS or FUS. Values represent means 6 SEM (n = 4). (E) FUS (5
mM) was
aggregated as in (C) for 60 min and processed for Thioflavin-T (ThT) fluorescence and compared to the ThT fluorescence of assembled Sup35-NM
fibers (5
mM monomer). Values represent means 6 SEM (n = 3). (F) FUS (5 mM) was aggregated as in (C) for 60 min. The amount of SDS-resistant FUS
was then determined and compared to the amount of SDS-resistant Sup35-NM in assembled Sup35-NM fibers (5
mM monomer). Values represent
means 6 SEM (n = 3).
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(Figure 8A,B). Rather, these proteins persist as small oligomeric
particles (Figure 8A,B). In the absence of TEV protease, both FUS
and FUS 1–422 did not aggregate but remained as small
oligomeric species (Figure 8C,D). After addition of TEV protease,
FUS and FUS 1–422 rapidly populated oligomeric forms, which
adopted a pore-like conformation reminiscent of pathological
oligomers formed by TDP-43, a-synuclein, and Ab42 (Figure 8E)
[15,42]. FUS 1–422 rapidly aggregated in an ordered manner to
generate separated filamentous structures (Figure 8C). Likewise,
full-length FUS also rapidly formed linear polymers (Figure 8D).
In both cases, these filaments were approximately 15–20 nm in
diameter and could extend several micrometers in length
(Figure 8C,D). Consistent with turbidity measurements, the
polymers formed by full-length FUS became tangled and stacked
against one another to form extremely large and complex
macroscopic networks (Figure 8D,F). FUS 1–422 polymers
remained more separated with limited lateral interaction
(Figure 8C,F). These ultrastructural observations explain why
FUS 1–422 aggregates are more difficult to detect by turbidity.
Importantly, the filamentous structures formed by both FUS
and FUS 1–422 bear striking resemblance to the FUS aggregates
observed in the degenerating motor neurons of ALS patients
[21,77]. In motor neurons of patients with juvenile ALS, FUS
forms filamentous aggregates with a uniform diameter of 15–20
nm, which are often associated with small granules [21,77]. The
filamentous structures formed by FUS and FUS 1–422 in isolation
(Figure 8C,D,F) are extremely similar to those observed in spinal
motor neurons in Figure 3C of Huang et al. [21]. In vitro, small
FUS or FUS 1–422 oligomers are often found clustered up against
the filamentous structures (Figure 8C,D,F). These oligomers may
correspond to the granular structures observed in association with
filamentous FUS aggregates in motor neurons of ALS patients
[21,77]. In sum, these observations suggest that in isolation FUS is
intrinsically capable of forming the aggregated structures observed
in motor neurons of ALS patients.
Taken together, the biochemical and EM data suggest that FUS
aggregation requires multiple domains in both N- and C-terminal
regions. Specifically, determinants in the N-terminal prion-like
domain (1–239) and the first C-terminal RGG domain (374–422)
are essential for the formation of filamentous structures. More C-
terminal regions (423–526) are then required for the formation of
large macroscopic aggregates detected by turbidity.
Figure 6. FUS aggregates more rapidly than TDP-43 in vitro. (A) GST-FUS or GST-TDP-43 (2.5 or 5 mM) was incubated in the presence of TEV
protease at 22uC for 0–90 min. Turbidity measurements were taken every minute to assess the extent of aggregation. A dataset representative of
three replicates is shown. (B) GST-FUS or GST-TDP-43 (2.5 or 5
mM) was incubated in the presence of TEV protease at 22uC with agitation (700 rpm) for
0–90 min. The extent of aggregation was determined by turbidity. A dataset representative of three replicates is shown.
doi:10.1371/journal.pbio.1000614.g006
Figure 7. Defining the domain requirements for the aggregation of pure FUS. (A) GST-FUS, GST-FUS 267–526, GST-FUS 1–373 (2.5 mMor
5
mM), or GST-FUS 1–422 (2.5 mM, 5 mM, or 20 mM) were incubated in the presence of TEV protease at 22uC for 0–90 min. Turbidity measurements
were taken every minute to assess the extent of aggregation. A dataset representative of three replicates is shown. (B) GST-FUS, GST-FUS 267–526,
GST-FUS 1–373, or GST-FUS 1–422 (5
mM) were incubated in the presence or absence of TEV protease at 22uC for 0–60 min. At the indicated times,
reactions were processed for sedimentation analysis. Pellet and supernatant fractions were resolved by SDS-PAGE and stained with Coomassie
Brilliant Blue. The amount of GST-FUS or FUS in the pellet fraction was determined by densitometry in comparison to known quantities of GST-FUS or
FUS. Values represent means 6 SEM (n = 3).
doi:10.1371/journal.pbio.1000614.g007
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Figure 8. Pure FUS aggregates resemble FUS aggregates in degenerating motor neurons of ALS patients. (A, B) GST-FUS 267–526
(2.5
mM) (A) or GST-FUS 1–373 (2.5 mM) (B) were incubated in the presence of TEV protease at 22uC for 0 or 60 min and processed for EM. Bar, 500 nm.
(C) GST-FUS 1–422 (2.5
mM) was incubated in the absence or presence of TEV protease at 22uC for 0–60 min. At the indicated times, reactions were
processed for EM. In the absence of TEV protease, very little aggregation occurs. In the presence of TEV protease, pore-shaped oligomers (arrows) and
filamentous polymers (arrowheads) rapidly assemble. At 60 min, the filamentous structures stay well separated but are sometimes associated with
smaller FUS 1–422 oligomers. Bar, 500 nm. (D) GST-FUS (2.5
mM) was incubated in the absence or presence of TEV protease at 22uC for 0–60 min. At
the indicated times, reactions were processed for EM. In the absence of TEV protease, very little aggregation occurs. In the presence of TEV protease,
pore-shaped oligomers (arrows) and filamentous polymers (arrowheads) rapidly assemble. The filamentous structures often form higher order
network structures by 30 and 60 min. (E) Gallery of pore-shaped FUS 1–422 oligomers formed after 30 min and pore-shaped FUS oligomers formed
after 10 min. Bar, 50 nm. (F) Lower magnification view of filamentous FUS 1–422 and FUS aggregates formed after 60 min in the presence of TEV
protease. Note that FUS aggregates accumulate as larger networks that conglomerate into large aggregates, whereas FUS 1–422 filaments remain
well separated. This difference in morphology likely explains why FUS aggregates generate a larger turbidity signal than FUS 1–422 aggregates. Bar,
500 nm.
doi:10.1371/journal.pbio.1000614.g008
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ALS-Linked FUS Mutations Do Not Affect Aggregation or
Toxicity
FUS mutations have been connected with some familial and
sporadic ALS cases [37]. We next used the yeast model to test the
effects of some of these mutations on FUS aggregation and toxicity
(Figure 9A). For TDP-43, we have used this approach to
determine that ALS-linked mutations increase TDP-43 aggrega-
tion and toxicity [15]. This increased toxicity of mutant TDP-43 in
yeast has been supported by independent studies in mammalian
cells and animal models [9,78–80]. To assess aggregation, we
expressed YFP-tagged fusions of WT FUS and 12 different ALS-
linked FUS mutants in yeast. These FUS variants were all
expressed at similar levels (Figure 9B). Moreover, comparison of
the number of proportion of yeast cells with three or more foci
revealed that ALS-linked FUS mutations do not promote FUS
aggregation in yeast (Figure 9C,D). Indeed, FUS aggregation was
slightly reduced in various ALS-linked FUS variants, although this
reduction was not statistically significant (Figure 9C,D). Consistent
with these observations, the ALS-linked FUS variants—H517Q,
R521C, and R521G—aggregated with very similar kinetics to WT
in pure protein aggregation assays, although aggregation was
slightly retarded in these mutants (Figure 9E). Collectively, these
data suggest that this set of ALS-linked FUS mutations, clustered
in the extreme C-terminal region of FUS, do not promote FUS
aggregation per se. Furthermore, we did not observe any
significant difference in toxicity between WT and ALS-linked
FUS variants (Figure 9F). These data are in contrast to TDP-43,
where several ALS-linked mutations promote aggregation and
toxicity [15].
It seems likely that in disease, these C-terminal ALS-linked FUS
mutations promote pathological events that are primarily upstream
of aggregation and toxicity. One obvious upstream event is
mislocalization to the cytoplasm. Indeed, studies in mammalian
cells suggest that ALS-linked FUS mutations can disrupt nuclear
import [52]. In yeast, FUS is already localized predominantly to the
cytoplasm (Figures 1C, 9C), so in this setting the ALS-linked
mutants are no more toxic than WT (Figure 9C,D,F). Thus, even
though FUS and TDP-43 are related RNA-binding proteins, the
mechanisms by which ALS-linked mutations contribute to disease
might be different for each protein [52]. Consequently, different
therapeutic strategies might be needed for FUS and TDP-43
proteinopathies. To examine this idea further, we performed two
genome-wide screens in yeast to (1) identify genetic modifiers of
FUS toxicity and (2) determine whether genetic modifiers of FUS
toxicity also affected TDP-43 toxicity.
A Yeas t Genome-Wide Overexpression Screen Identifies
Modifiers of FUS Toxicity
Of the many experimental benefits afforded by the yeast system
[13], the chief advantage is the ability to perform high-throughput
genetic modifier screens. Therefore, to provide insight into cellular
mechanisms underpinning FUS toxicity, we performed two
unbiased yeast genetic modifier screens to identify genes that
enhance or suppress FUS toxicity. We reasoned that the genes
identified by these screens would illuminate cellular pathways
perturbed by abnormal FUS accumulation and suggest potential
novel targets for therapeutic intervention. Similar approaches have
elucidated modifiers of the Parkinson’s disease protein a-synuclein
[39,40,45,81], a mutant form of the Huntington’s disease protein
huntingtin [44,45], and more recently, the ALS protein TDP-43
([16]; A. Elden and A.D.G. unpublished). In the latter example,
the yeast system allowed definition of a common genetic risk factor
for ALS in humans [16].
First, we performed a plasmid overexpression screen (Figure 10A).
We individually transformed 5,500 yeast genes, which comprise the
Yeast FLEXGene plasmid overexpresssion library [82], into a yeast
strain harboring an integrated galactose-inducible FUS expression
plasmid. We then identified yeast genes that suppressed or
enhanced FUS toxicity when overexpressed (Figure 10B). We
repeated the screen three independent times and only selected hits
that reproduced all three times. Genes from the screen that
enhanced FUS toxicity, but also caused toxicity when overexpressed
in WT yeast cells, were eliminated because these were unlikely to be
specific to FUS. We also eliminated certain genes involved in
carbohydrate metabolism or galactose-regulated gene expression
because, based on previous screens with this library, we have found
that they simply affect expression from the galactose-regulated
promoter and are unlikely to relate to FUS biology. Indeed, most of
these were also recovered as hits in screens with a galactose-
regulated toxic huntingtin, a-syn or TDP-43 ([16,39,44,45,81]; A.
Elden and A.D.G. unpublished). Finally, we retested 10 random
plasmids (six suppressors and four enhancers) by transforming them
into a fresh yeast strain harboring the integrated FUS expression
plasmid and performed spotting assays and all 10 of these were
confirmed (Figure S5).
Following the above validation and filtering procedures, we
identified 24 genes that suppressed and 10 genes that enhanced FUS
toxicity when overexpressed (Table 1). The largest functional class
enriched in the screen included RNA-binding proteins and proteins
involved in RNA metabolism (Figure 10C). Thus, RNA metabolic
pathways play a key role in FUS pathogenesis. Importantly, of 71
genes from this library that modify a-synuclein toxicity in yeast
[39,40], only two (Cdc4 and Tps3) affected FUS toxicity. This lack of
overlap underscores the specificity of the screen for FUS biology and
pathobiology. Moreover, this specificity indicates that the screen
does not simply identify generic cellular responses to misfolded
proteins. Even more remarkably, out of the 40 yeast genes that we
have found to modify TDP-43 toxicity when overexpressed ([16] and
A. Elden and A.D.G. unpublished observations), only two (Fmp48
and Tis11) affected FUS toxicity. Thus, despite being similar RNA-
binding proteins, the mechanisms by which FUS and TDP-43
contribute to disease are likely to be very different.
Several of the yeast genes that modified FUS toxicity have
human homologs. Thus, pathways involved in FUS toxicity in
yeast are likely conserved to man. Interestingly, FUS has recently
been shown to co-localize with stress granules in transfected cells
[51,52]. Furthermore, cytoplasmic FUS-positive inclusions in ALS
and FTLD-U patients contain stress granule markers [51,52].
Stress granules and P-bodies are transient cytoplasmic structures
containing RNAs and RNA binding proteins, including translation
initiation factors and the polyA-binding protein (PABP-1), which
are sites where cells sequester mRNAs, during situations of stress,
to inhibit translation initiation [83]. Notably, we identified two
translation initiation factors (Tif2 and Tif3) and Pab1, the yeast
homolog of human PABP-1, which is involved in stress granule
assembly in yeast, as suppressors of FUS toxicity (Table 1). Thus,
in addition to being markers of FUS-positive inclusions in disease,
stress granule components might play an important role in
mediating FUS toxicity. Approaches aimed at manipulating stress
granule assembly might be an effective therapeutic approach.
Overexpression Suppressors Isolated from Yeast Also
Suppress FUS Toxicity in Mammalian Cells
As an initial step to extend our findings from yeast to
mammalian cells, we selected genes from our overexpression
screen for further analysis in a mammalian cell culture FUS
toxicity model. We tested two distinct suppressor genes, FBXW7
Mechanisms of FUS Aggregation and Toxicity
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Figure 9. The effect of ALS-linked FUS mutations on aggregation and toxicity. (A) Diagram indicating disease-associated FUS mutations
tested in this study. (B) Immunoblot showing equivalent expression levels of WT or mutant FUS. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as a loading control. (C) ALS-linked mutations did not significantly affect FUS aggregation in yeast. (D) The effect of ALS-linked FU S
mutations on aggregation in yeast cells was quantified by counting the number of cells containing .3 FUS-YFP foci (as in [15] for TDP-43). Values
represent means 6 SEM (n$3, at least 200 cells per sample). As for FUS toxicity, these ALS-linked mutations did not significantly enhance FUS
aggregation, in contrast to TDP-43 mutations, which did increase aggregation [15]. (E) GST-FUS, GST-FUS H517Q, GST-FUS R521H, or GST-FUS R521C
(2.5
mM) was incubated in the presence of TEV protease at 22uC for 0–90 min. Turbidity measurements were taken every minute to assess the extent
of aggregation. A dataset representative of three replicates is shown. (F) Spotting assay to compare the toxicity of WT and mutant FUS. Serial
dilutions of yeast cells transformed with galactose-inducible empty vector, WT, or mutant FUS-YFP constructs. Transformants were spotted on
glucose (non-inducing) or galactose (inducing) containing agar plates, and growth was assessed after 3 d. In contrast to TDP-43 [15], the ALS-linked
FUS mutations did not enhance FUS toxicity in yeast.
doi:10.1371/journal.pbio.1000614.g009
Mechanisms of FUS Aggregation and Toxicity
PLoS Biology | www.plosbiology.org 14 April 2011 | Volume 9 | Issue 4 | e1000614
and EIF4A1, which are the human homologs of yeast Cdc4 and
Tif2, respectively (Table 1). We transfected HEK293T cells with
WT FUS or two ALS-linked FUS mutants, R521C and R521H.
The FUS mutants were more toxic than WT FUS, which only
slightly reduced viability (Figure 10D). Co-transfection with
FBXW7 or EIF4A1 suppressed toxicity of WT FUS as well as
the ALS-linked FUS mutants (Figure 10D). Similar results were
observed in COS-7 cells (unpublished data). The FUS toxicity
modifier genes and pathways identified in our yeast screens will
have to be validated in neuronal cells and eventually animal
models. However, the ability of FBXW7 and EIF4A1 to suppress
toxicity in human cells, which are separated from yeast by ,1
billion years of evolution, provides evidence that highly conserved
genetic interactions involving FUS, discovered in yeast, can be
highly relevant to mammalian cells.
A Yeast Genome-Wide Deletion Screen Identifies
Modifiers of FUS Toxicity
To complement the yeast overexpression screen, we also
performed a deletion screen. The yeast genome contains ,6,000
Figure 10. Yeast plasmid overexpression screen identifies suppressors and enhancers of FUS toxicity. (A) Schematic of yeast genetic
screen. Yeast cells harboring an integrated galactose-inducible FUS-YFP cassette were individually transformed with a library of 5,500 yeast open
reading frames (ORFs) and spotted onto galactose plates to induce expression of FUS and each gene from the library. (B) A representative plate from
the yeast screen. Each spot represents a yeast strain expressing FUS along with one gene from the library. Examples of genes that suppressed FUS
toxicity (improved growth) are indicated by green arrows and enhancers of toxicity (inhibited growth) are indicated by red arrows. (C) A histogram
indicating the functional categories of genes enriched as hits in the screen compared to the yeast genome. Genes involved in transcription and RNA
metabolism were significantly overrepresented as hits in the screen (indicated by *). (D) Human homologs of two FUS toxicity modifier genes from
the yeast screen, FBXW7 and EIF4A1, suppressed FUS toxicity in human cells (HEK293T), when co-transfected with FUS or ALS-linked FUS mutants,
R521C and R521H. Cell viability was assessed by MTT assay. Values represent means 6 S.D. (n = 3).
doi:10.1371/journal.pbio.1000614.g010
Mechanisms of FUS Aggregation and Toxicity
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yeast genes and ,4,850 of these are non-essential [84,85]. We
used synthetic genetic array (SGA) analysis [86,87] to introduce a
FUS expression plasmid into each non-essential yeast deletion
strain by mating (Figure 11A). Following sporulation, we
selectively germinated meiotic progeny containing both the FUS
plasmid and the gene deletion. We compared growth of each
strain on glucose (FUS expression ‘‘off’’) to that on galactose (FUS
expression ‘‘on’’). We identified some yeast deletions that
enhanced FUS toxicity (aggravating interaction) and others that
suppressed toxicity (alleviating interaction) (Figure 11B). As for the
overexpression screen, we repeated the deletion screen three
independent times and only selected hits that reproduced all three
times and filtered out deletion strains that grew poorly on
galactose-containing media, even in the absence of FUS (using
published data on yeast deletion strain fitness on galactose and in
house measurements of the yeast deletion collection grown on
galactose). Genetic interactions were further confirmed by random
spore analysis and the integrity of the deletions verified by
sequencing the deletion specific bar codes. We also independently
confirmed six random hits by remaking the deletions, confirming
the deletions by PCR, and then transforming those deletion strains
with the FUS expression plasmid and performing spotting assays.
We indentified 36 deletions that suppressed FUS toxicity and 24
that enhanced toxicity (Table 2). Deletions of yeast genes involved
Table 1. Yeast genes that suppress or enhance FUS toxicity when overexpressed.
Effect Gene Human Homolog Function
Suppressor CDC4 FBXW7 F-box protein required for G1/S and G2/M transition
Suppressor CUE2 Protein of unknown function
Suppressor ECM32 UPF1 DNA dependent ATPase/DNA helicase, involved in modul ating translation termination
Suppressor EDC3 Non-essential conserved protein of unknown function, plays a role in mRNA decapping
Suppressor FHL1 Putative transcriptional regulator, required for rRNA processing
Suppressor FMP48 STK36 Mitochondrial protein of unknown function
Suppressor NAM8 TRNAU1AP RNA binding protein, component of the U1 snRNP protein
Suppressor PAB1 PABPC4 Poly(A) binding protein, part of the 39-end RNA-processing complex, involved in stress
granule formation
Suppressor PIG1 Putative targeting subunit for the type-1 protein phosphatase Glc7p
Suppressor SBP1 Nucleolar single-strand nucleic acid binding protein, associates with small nuclear RNAs
Suppressor SEY1 Protein of unknown function, contains two predicted GTP-binding motifs
Suppressor SKO1 Basic leucine zipper (bZIP) transcription factor of the ATF/CREB family
Suppressor SYN8 STX8 Endosomal SNARE related to mammalian syntaxin 8
Suppressor TIF2 EIF4A1 Translation initiation factor eIF4A, RNA helicase that couples ATPase activity to RNA
binding and unwinding, involved in stress granule formation
Suppressor TIF3 EIF4B Translation initiation factor eIF-4B, has RNA annealin g activity, contains an RNA
recognition motif and binds to single-stranded RNA, involved in stress granule formation
Suppressor TIS11 mRNA-binding protein involved in iron homeostasis
Suppressor TPS3 Regulatory subunit of trehalose-6-phosphate synthase/phosphatase complex
Suppressor TRM11 TRMT11 Catalytic subunit of an adoMet-dependent tRNA methyltransferase complex
Suppressor VHR1 Transcriptional activator
Suppressor YHR151C Unknown
Suppressor YOR062C Unknown
Suppressor YPR147C C2orf43 Unknown
Suppressor ZDS2 Protein that interacts with silencing proteins at the telomere, involved in transcriptional
silencing
Enhancer CLB2 CCNB1 B-type cyclin involved in cell cycle progression
Enhancer CST6 Basic leucine zipper (bZIP) transcription factor of the ATF/CREB family
Enhancer FZO1 Mitochondrial integral membrane protein involved in mitochondrial fusion and
maintenance of the mitochondrial genome
Enhancer HOF1 Bud neck-localized protein required for cytokinesis
Enhancer INM1 IMPA1 Inositol monophosphatase
Enhancer IRC3 EIF4A3 Putative RNA helicase of the DEAH/D-box family
Enhancer NAB3 Single stranded RNA binding protein, required for termination of non-poly(A) transcripts
and efficient splicing
Enhancer PET111 Specific translational activator for the COX2 mRNA, located in the mitochondrial inner
membrane
Enhancer TRM5 TRMT5 tRNA methyltransferase
Enhancer YMR166C SLC25A26 Predicted transporter of the mitochondrial inner membrane
doi:10.1371/journal.pbio.1000614.t001
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Figure 11. Yeast deletion screen identifies suppressors and enhancers of FUS toxicity. (A) Schematic of yeast deletion screen, based on
[87]. The galactose-inducible FUS expression construct (pAG416Gal-FUS-YFP) was introduced into MATa strain Y7092 to generate the query strain.
This query strain was mated to the yeast haploid deletion collection of non-essential genes (MATa, each gene deleted with KanMX cassette (confers
resistance to G418)). Mating, sporulation, and mutant selection were performed using a Singer RoToR HDA (Singer Instruments, Somerset, UK).
Haploid mutants harboring the FUS expression plasmid were grown in the presence of glucose (FUS expression ‘‘off’’) or galactose (FUS expression
‘‘on’’). Following growth at 30uC for 2 d, plates were photographed and colony sizes measured by ImageJ image analysis software, based on [104]. (B)
A representative plate from the deletion screen. Left is glucose (deletion alone, e.g. xxxD) and right is galactose (deletion + FUS expression, e.g. xxxD
+ FUS). Each plate contains 384 different strains pinned in duplicate (768 total). The red arrows point to an aggravating genetic interaction (toxicity
Mechanisms of FUS Aggregation and Toxicity
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in RNA metabolic processes, ribosome biogenesis, and cellular
stress responses were enriched as hits (Figure 11C). Many of these
genes have human homologs (Table 2). One interesting deletion
suppressor was Sse1, a member of the Hsp70 chaperone family,
which promotes Sup35 prion formation [88] and might also
promote FUS aggregation. Two other notable deletion suppressors
were Pub1 (TIAL1 in human) and Lsm7 (LSM7 in human),
components of stress granules and P-bodies, respectively. Further-
more, TIAL1 (and Pub1) contains a prion-like domain [32,89],
which can template the aggregation of the polyQ protein
huntingtin [90], suggesting that FUS aggregation and cytoplasmic
sequestration might be templated by similar mechanisms [24,91].
Again, as for the plasmid overexpression screen, genetic
manipulations that affect stress granule components are sufficient
to mitigate FUS toxicity. And, as for the overexpression screen,
there was little overlap between the FUS and TDP-43 modifier
genes. In a broader sense, the collection of deletion suppressors of
FUS toxicity is an interesting class, because these could represent
attractive therapeutic targets for small molecule inhibitors or RNA
interference. Taken together, the genetic modifiers uncovered by
the yeast overexpression and deletion screens provide insight into
the pathways affected by FUS. The way is now open to develop
therapeutic strategies that target these pathways.
Discussion
We have established a pure protein aggregation assay and a
yeast model to gain insight into how FUS contributes to disease
pathogenesis. We have recently used a similar approach to define
mechanisms underpinning TDP-43 aggregation and toxicity [14],
as well as the pathogenic mechanism of ALS-linked TDP-43
mutants [15]. Using the yeast system we have also identified potent
modifiers of TDP-43 toxicity [16]. One such modifier is ataxin 2,
which can harbor intermediate-length polyQ expansions that are
associated with increased risk for ALS in humans [16]. Like TDP-
43, we find that, in isolation, FUS is an intrinsically aggregation-
prone protein. FUS rapidly assembles into pore-like oligomeric
species and filamentous structures that closely resemble the
ultrastructure of FUS aggregates in degenerating motor neurons
of ALS patients. Thus, all the information needed to assemble
these structures is encoded in the primary sequence of FUS. Like
TDP-43, expression of FUS in yeast results in cytoplasmic FUS
aggregation, colocalization of these inclusions with stress granules
and toxicity, modeling key features seen in human disease
[17,18,21,23,52]. In further similarity to TDP-43, disabling the
RNA binding activity of FUS reduced toxicity. Thus, we propose
that the misfolded forms of FUS likely cause toxicity by binding to
and sequestering essential RNAs or perhaps by interfering with the
normal shuttling, stability, or metabolism of RNA. Importantly,
FUS immunoreactive cytoplasmic inclusions now appear to
characterize ALS and FTLD broadly, not only rare cases linked
to FUS mutations [21,23,92]. Together these advances make it
clear that FUS is a key aggregated protein in ALS, just as a-
synuclein is in Parkinson’s disease and huntingtin is in Hunting-
ton’s disease [33].
Despite these similarities, we have uncovered key differences in
the regions of the proteins that dictate aggregation and toxicity.
For TDP-43, pure protein data and results from yeast and other
model systems suggest that the C-terminal prion-like domain
(Figure 1A) [33] plays a major role in driving aggregation
[14,15,66,93]. For FUS, we find that the N-terminal region,
containing a predicted prion-like domain (Figure 1A) [33], is also
important for aggregation in vitro and for aggregation and toxicity
in yeast cells. However, C-terminal regions in FUS, particularly
the first RGG domain, are also critical. Intriguingly, the first RGG
domain also contains a short region (amino acids 391–407) that is
detected by an algorithm designed to isolate prion-like domains
[32,33] but does not quite reach significance (Figure S1). The
requirement for two specific, disparate portions of FUS for the
ordered formation of filamentous structures raises the possibility
that communication between the N-terminal prion-like domain
(amino acids 1–239) and first RGG domain (amino acids 374–422)
might mediate a self-organizing assembly process. This process
might even involve an intermolecular domain swap: a common
mechanism that usually involves domains at the N- and C-
terminal ends of proteins and can promote the polymerization of
filamentous structures in various designed and natural proteins
[94–96]. Thus, strategies aimed at targeting either the appropriate
N- or C-terminal portions of FUS could be effective at mitigating
FUS aggregation in disease. Indeed, our in vitro and yeast models
could open up new therapeutic avenues and provide the basic
screening system to isolate specific molecules able to antagonize
and reverse FUS aggregation and toxicity.
With regard to toxicity, the minimal toxic FUS fragment
comprises the N-terminal prion-like domain, RRM, and the first
RGG domain (1–422). These findings contrast with TDP-43,
where the prion-like domain plus RRM2 are sufficient to drive
aggregation and toxicity [14]. Indeed, a proteolytic fragment
corresponding to these portions of TDP-43 is a pathogenic
signature of ALS and FTLD-TDP [3]. By contrast, a similar
pathogenic FUS fragment has not been identified in ALS or
FTLD-FUS patients, which likely reflects the fact that the
equivalent regions of FUS (1–373) are insufficient for aggregation
and toxicity.
Mutations in the C-terminal domains of FUS and TDP-43 have
both been linked to ALS [6,37]. Interestingly, whereas some ALS-
linked mutations in TDP-43 can increase stability, aggregation,
cytoplasmic accumulation, and toxicity in yeast, mammalian cells,
and animal models [15,16,78,80,97], the mechanisms by which
FUS mutations contribute to disease appear to be distinct. Our
results in yeast and with pure protein show that C-terminal FUS
mutations do not promote aggregation per se. Instead of
enhancing aggregation, these mutations, especially those in the
extreme C-terminal region of the protein (amino acids 502–526),
disrupt a NLS, leading to increased cytoplasmic accumulation of
FUS [52]. Interestingly, the severity of the effects of the mutations
on FUS localization in cells correlate well with age of onset of ALS
in humans, with stronger mutations resulting in earlier disease
onset and more cytoplasmic FUS accumulation [52]. These results
suggest distinct mechanisms by which ALS-linked FUS and TDP-
43 mutations contribute to disease.
Despite these differences, both TDP-43 and FUS have been
shown to re-localize to stress granules and P-bodies, transient sites
of RNA processing that assemble during cellular stress or injury
and are conserved from yeast to man [59,60,62,98]. Both TDP-43
and FUS have been purified in a complex with one another and
enhancer), in which the gene deletion + FUS grows slower than FUS or the deletion alone. The green arrows point to an alleviating genetic
interaction (toxicity suppressor), in which the gene deletion + FUS grows better than FUS or the deletion alone. (C) A histogram indicating the
functional categories of genes enriched as hits in the screen compared to the yeast genome. Genes involved in RNA metabolism, ribosome
biogenesis, and cellular stress responses were significantly overrepresented as hits in the screen (indicated by *).
doi:10.1371/journal.pbio.1000614.g011
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Table 2. Yeast genes that suppress or enhance FUS toxicity when deleted.
Effect Gene Human Homolog Function
Suppressor ALF1 TBCB Alpha-tubulin folding protein
Suppressor BUD26 Dubious open reading frame
Suppressor CGI121 TPRKB Protein involved in telomere uncapping and elongation
Suppressor CLB2 CCNB1 B-type cyclin involved in cell cycle progression
Suppressor FYV7 Essential protein required for maturation of 18S rRNA
Suppressor GIS2 ZCCHC13 Protein proposed to be involved in the RAS/cAMP signaling pathway
Suppressor HIT1 Protein of unknown function, required for growth at high temperature
Suppressor HMO1 Chromatin associated high mobility group (HMG) family member involved in
genome maintenance
Suppressor IPK1 Inositol 1,3,4,5,6-pentakisphosphate 2-kinase
Suppressor LSM7 LSM7 Lsm (Like Sm) protein, part of heteroheptameric complexes mRNA decayor in
processing tRNA, snoRNA, and rRNA, involved in stress granul e formation
Suppressor LTV1 Component of the GSE complex, which is required for proper sorting of amino
acid permease Gap1p
Suppressor MFT1 Subunit of the THO complex, which is a nuclear complex involved in
transcription elongation and mitotic recombination
Suppressor MRT4 MRTO4 Protein involved in mRNA turnover and ribosome assembly, localizes to the
nucleolus
Suppressor NOP16 Constituent of 66S pre-ribosomal particles, involved in 60S ribosomal subunit
biogenesis
Suppressor NPR2 TUSC4 Component of an evolutionarily conserved Npr2/3 complex that mediates
downregulation of TORC1 activity in response to amino acid limitation
Suppressor NSR1 NCL Nucleolar protein that binds nuclear localization sequences, required for pre-
rRNA processing and ribosome biogenesis
Suppressor NUP84 NUP107 Subunit of the nuclear pore complex (NPC), plays a role in nuclear mRNA export
and NPC biogenesis
Suppressor PPM1 Carboxyl methyltransferase, methylates the C terminus of the protein
phosphatase 2A catalytic subunit
Suppressor PUB1 TIAL1 Poly (A)+ RNA-binding protein, component of glucose deprivation induced
stress granules, involved in P-body-dependent granule assembly
Suppressor RAD50 RAD50 Subunit of MRX complex, involved in processing double-strand DNA breaks in
vegetative cells
Suppressor RPL14A RPL14 Component of the large (60S) ribosomal subunit
Suppressor RPL19B RPL19 Component of the large (60S) ribosomal subunit
Suppressor RPP2B RPLP2 Ribosomal protein P2 beta, a component of the ribosomal stalk
Suppressor RPS10A RPS10L Component of the small (40S) ribosomal subunit
Suppressor RPS6B RPS6 Component of the small (40S) ribosomal subunit
Suppressor RPS8A RPS8 Component of the small (40S) ribosomal subunit
Suppressor SSE1 HSPA4 Hsp70 ATPase that is a component of the heat shock protein Hsp90 chaperone
complex, nucleotide exchange factor for Ssa1
Suppressor THP2 Subunit of the THO complex and TREX complex, involved in telomere
maintenance
Suppressor TSR2 TSR2 Protein with a potential role in pre-rRNA processing
Suppressor VPS64 Protein required for cytoplasm to vacuole targeting of proteins
Suppressor YDR417C Dubious open reading frame, partially overlaps the verified ORF RPL12B/
YDR418W
Suppressor YGL072C Dubious open reading frame, partially overlaps the verified gene HS F1
Suppressor YGL088W Dubious open reading frame, partially overlaps snR10, a snoRNA required for
preRNA processing
Suppressor YGL165C Dubious open reading frame, partially overlaps the verified ORF CUP2/YGL166W
Suppressor YNR005C Dubious open reading frame
Suppressor YOR309C AL138690.1 Dubious open reading frame, partially overlaps the verified gene NOP58
Enhancer ATP5 ATP50 Subunit 5 of the stator stalk of mitochondrial F1F0 ATP synthase
Enhancer CBT1 Protein involved in 59 end processing of mitochondrial COB, 15S_rRNA, and
RPM1 transcripts
Mechanisms of FUS Aggregation and Toxicity
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with various components of the RNA processing machinery,
including stress granules and P-bodies [62,97]. Moreover, stress
granule markers, including PABP-1, are present in disease-
associated cytoplasmic FUS accumulations [52]. ALS-linked
FUS mutants appear more prone to entering stress granules
[51]. However, it remains unclear whether stress granule assembly
contributes to FUS toxicity or is simply a downstream conse-
quence of cellular stress associated with degeneration. Our
identification of several key P-body and stress granule components
as potent genetic modifiers of FUS toxicity suggests a mechanistic
connection that, if validated in animal models, represents a
potentially tractable new therapeutic angle. We also note that for
the majority of overexpression or deletion suppressors that we
have examined so far, we do not see a major difference in FUS
aggregation. This suggests that these genes act downstream or in
parallel to FUS aggregation. Alternatively, these modifiers may
affect FUS aggregation (e.g., composition or dynamics of FUS
inclusions) in subtle ways that we have so far not been able to
visualize.
Curiously, there was a conspicuous lack of overlap between
genetic modifiers of FUS toxicity and TDP-43 toxicity. These
genetic data suggest two interesting possibilities. On one hand,
targeting the modifiers in common between TDP-43 and FUS
might have broad therapeutic utility for ALS. On the other hand,
defining the key differences between FUS and TDP-43 pathogenic
mechanisms will empower a more accurate understanding of how
these seemingly similar proteins might contribute to disease in
different ways.
What is the connection between TDP-43, FUS, and ALS? Does
each protein contribute separately to the disease, or do they share
a common disease pathway? The lack of overlap in genetic
modifiers suggests that the precise mechanism of TDP-43 and
FUS toxicity may be subtly different. Moreover, initial reports
suggested FUS cytoplasmic accumulations were specific to rare
cases of ALS, owing to FUS mutations, and that these inclusions
were devoid of TDP-43 aggregates [17]. However, in one study,
using optimized antigen-unmasking methods, FUS cytoplasmic
immunoreactivity has recently been detected broadly in sporadic
and familial ALS, including cases with TDP-43 aggregates, as well
as cases without FUS mutations [92]. Further, FUS and TDP-43
have been found to physically associate in a complex [97],
indicating that both TDP-43 and FUS, even in the WT state, likely
Effect Gene Human Homolog Function
Enhancer COX5A COX5A Subunit Va of cytochrome c oxidase
Enhancer EAF1 Component of the NuA4 histone acetyltransferase complex
Enhancer FUM1 FH Fumarase, converts fumaric acid to L-malic acid in the TCA cycle
Enhancer GCN4 Basic leucine zipper transcriptional activator of amino acid biosynthetic genes in
response to amino acid starvation
Enhancer KGD2 DLST Dihydrolipoyl transsuccinylase, component of the mitochondrial alpha-
ketoglutarate dehydrogenase complex
Enhancer MAK32 Protein necessary for structural stability of L-A double-stranded RNA-containing
particles
Enhancer MRP13 Mitochondrial ribosomal protein of the small subunit
Enhancer MRP49 Mitochondrial ribosomal protein of the large subunit, not essential for
mitochondrial translation
Enhancer MRPL39 Mitochondrial ribosomal protein of the large subunit
Enhancer MSS1 GTPBP3 Mitochondrial protein, involved in the 5-carboxymethylaminomethyl
modification of the wobble uridine base in mitochondrial tRNAs
Enhancer OCA1 Putative protein tyrosine phosphatase, required for cell cycle arrest in response
to oxidative damage of DNA
Enhancer REC102 Protein involved in early stages of meiotic recombination
Enhancer RIM15 MAST1 Glucose-repressible protein kinase involved in signa l transduction during cell
proliferation in response to nutrients
Enhancer RTT103 RPRD1A Protein that interacts with exonuclease Rat1p and Rai1p and plays a role in
transcription termination by RNA polymerase II
Enhancer SLM3 TRMU tRNA-specific 2-thiouridylase, responsible for 2-thiolation of the wobble base of
mitochondrial tRNAs
Enhancer SLT2 UHMK1 Serine/threonine MAP kinase involved in regulating the maintenance of cell wall
integrity and progression through the cell cycle
Enhancer TBS1 Putative protein of unknown function
Enhancer YDL032W Dubious open reading frame unlikely to encode a protein, partially overlaps
verified gene SLM3/YDL033C
Enhancer YDR049W ANKZF1 Zinc finger protein, putative transcription factor that may interact with proteins
involved in histone acetylation or deacetylation
Enhancer YDR248C C9orf103 Putative protein of unknown function
Enhancer YER128W Putative protein of unknown function
Enhancer YLR218C Protein that localizes to the mitochondrial intermembrane space
doi:10.1371/journal.pbio.1000614.t002
Table 2. Cont.
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contribute broadly to ALS pathogenesis. Therefore, defining
mechanisms by which WT versions of these proteins are toxic to
cells, as we report here for FUS and in previous studies for TDP-
43 [14–16], will likely be informative to not only rare familial cases
but to the much more common sporadic forms as well.
The discovery of RNA-bindingproteinsTDP-43andFUSin
ALS has re-invigorated the focus on RNA processing pathways
in ALS [5,37,99]. Our identification o f potent gen etic modifiers
of FUS toxicity in yeast, including a large number of conserved
RNA metabolism genes, as well as key stress granule
components, will provide a toehold for future studies aimed at
elucidating the mechanisms by which FUS interfaces with these
RNA p rocessing pathways in disease. However, our study also
suggests caution in assuming, b ased on sequence and structural
similarity, that both TDP-43 and FUS contribute to disease via
the same or similar mechanisms [38]. While there are clear
similarities between the two proteins, there are also important
differences, which we have defined here. Furthermore, the fact
that genetic modifiers uncovered in screens for TDP-43 and
FUS proteotoxicty are surprisingly distinct argues further that
there are likely dif ferent underlying pathogen ic mechanisms for
FUS and TDP-43 proteinopathies. This conceptual framework
we have established will aid the development o f novel
therapeutic approaches.
Materials and Methods
Yeast Strains, Media, and Plasmids
Yeast cells were grown in rich media (YPD) or in synthetic
media lacking uracil and containing 2% glucose (SD/-Ura),
raffinose (SRaf/-Ura), or galactose (SGal/-Ura).
A FUS Gateway entry clone was obtained from Invitrogen,
containing full-length human FUS in the vector pDONR221. A
Gateway LR reaction was used to shuttle FUS into Gateway-
compatible yeast expression vectors (pAG vectors, [100], http://
www.addgene.org/yeast_gateway). To generate C-terminally YFP-
tagged FUS constructs, a two-step PCR protocol was used to
amplify FUS (or truncated versions) without a stop codon and
incorporate the Gateway attB1 and attB2 sites along with a Kozak
consensus sequence. Resulting PCR products were shuttled into
pDONR221 using a Gateway BR reaction. The entry clones
(FUS
nostop
) were then used in LR reactions with pAG426Gal-ccdB-
YFP to generate the 2 micron FUS-YFP fusion constructs and
pAG416Gal-ccdB-YFP to generate the CEN FUS-YFP constructs.
Primer sequences are available upon request. To generate the
integrating FUS construct, the FUS entry clone was used in an LR
reaction with pAG303Gal-ccdB. Expression constructs for TDP-43
have been described previously [14,15].
ALS-linked point mutations, based on [38], were introduced into
FUS using the QuickChange Site-Directed Mutagenesis Kit
(Agilent) according to the manufacturer’s instructions. Mutations
were verified by DNA sequencing. To disable FUS RNA binding,
we mutated four conserved phenylalanine residues (aa 305, 341,
359, 368) within the FUS RNA recognition motif (RRM) to leucine.
Two micron plasmid constructs (e.g., pAG426Gal-FUS-YFP)
were transformed into BY4741 (MATa his3 leu2 met15 ura3). The
FUS integrating strain was generated by linearizing pAG303Gal-
FUS by Nhe I restriction digest, followed by transformation into
the w303 strain (MATa can1-100, his3-11,15, leu2-3,112, trp1-1,
ura3-1, ade2-1).
To introduce the SV40 NLS to the N-terminus of FUS, we used
PCR, incorporating DNA sequences encoding the SV40 NLS
(PPKKKRKV), optimized for yeast translation (CCA CCA AAA
AAA AAA AGA AAA GTT) into the forward primer, following a
start codon (ATG) and in frame with FUS. We verified the
construct by DNA sequencing.
Yeast Transformation and Spotting Assays
Yeast procedures were performed according to standard
protocols [101]. We used the PEG/lithium acetate method to
transform yeast with plasmid DNA [102]. For spotting assays,
yeast cells were grown overnight at 30uC in liquid media
containing raffinose (SRaf/-Ura) until they reached log or mid-
log phase. Cultures were then normalized for OD600, serially
diluted and spotted onto synthetic solid media containing glucose
or galactose lacking uracil and were grown at 30uC for 2–3 d.
Immunoblotting
Yeast lysates were subjected to SDS/PAGE (4%–12% gradient,
Invitrogen) and transferred to a PVDF membrane (Invitrogen).
Membranes were blocked with 5% nonfat dry milk in PBS for 1 h
at room temperature. Primary antibody incubations were
performed overnight at 4uC or at room temperature for 1–2 h.
After washing with PBS, membranes were incubated with a
horseradish peroxidase-conjugated secondary antibody for 1 h at
room temperature, followed by washing in PBS+0.1% Tween 20
(PBST). Proteins were detected with Immobilon Western HRP
Chemiluminescent Substrate (Millipore). Primary antibody dilu-
tions were as follows: anti-GFP monoclonal antibody (Roche),
1:5,000; Phosphoglycerate Kinase 1 (PGK1) antibody (Invitrogen),
1:500; glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
1:5,000; FUS rabbit polyclonal antibody (Bethyl), 1:10,000.
HRP-conjugated anti-mouse and anti-rabbit secondary antibodies
were used at 1:5,000.
Fluorescence Microscopy
For fluorescence microscopy experiments, single colony isolates
of the yeast strains were grown to mid-log phase in SRaf/-Ura
media at 30uC. Cultures were spun down and resuspended in the
same volume of SGal/-Ura to induce expression of the FUS
constructs. Cultures were induced with galactose for 4–6 h before
being stained with DAPI to visualize nuclei and processed for
microscopy. Images were obtained using an Olympus IX70
inverted microscope and a Photometrics CoolSnap HQ 12-bit
CCD camera. Z-stacks of several fields were collected for each
strain. The images were deblurred using a nearest neighbor
algorithm in the Deltavision Softworx software and representative
cells were chosen for figures.
Quantification of FUS Aggregation in Yeast
To assess differences in aggregation between wild-type and
mutant FUS, yeast cultures were grown, induced, and processed as
described above after having normalized all yeast cultures to
OD
600nm
= 0.2 prior to galactose induction. After 6 h of induction,
the identities of the samples were blinded to the observer before
being examined. Several fields of cells were randomly chosen using
the DAPI filter to prevent any bias towards populations of cells
with increased amounts of aggregation in addition to obtaining the
total number of cells in any given field. At least 200 cells per
sample were counted for each replicate. Only cells with greater
than three foci under the YFP channel were considered as cells
with aggregating FUS.
Yeast Plasmid Overexpression Screen
Plasmids of 5,500 full-length yeast ORFs (Yeast FLEXGene
collection, [82]) were dried in individual wells of 96-well microtiter
plates and transformed into a strain expressing FUS integrated at
Mechanisms of FUS Aggregation and Toxicity
PLoS Biology | www.plosbiology.org 21 April 2011 | Volume 9 | Issue 4 | e1000614
the HIS3 locus. A standard lithium acetate transformation protocol
was modified for automation and used by employing a
BIOROBOT Rapidplate 96-well pipettor (Qiagen). The transfor-
mants were grown in synthetic deficient media lacking uracil (SD-
Ura) with glucose. 48 h later, the cultures were inoculated into
fresh SRaf-Ura media and allowed to reach stationary phase.
Then the cells were spotted on to SD-Ura + glucose and SD-Ura +
galactose agar plates. Suppressors and enhancers of FUS were
identified on galactose plates after 2–3 d of growth at 30uC. The
entire screen was repeated three times and only hits that
reproduced all three times were selected for further validation.
Toxicity enhancers were further tested in WT yeast cells to
eliminate genes that were simply toxic when overexpressed.
Immunoblotting was performed to test all modifiers for their effect
on FUS expression.
Yeast Deletion Screen
This screen was performed as described in [86,87,103], with
some modifications, using a Singer RoToR HDA (Singer
Instruments, Somerset, UK). The galactose-inducible FUS
expression construct (pAG416Gal-FUS-YFP) was introduced into
MATa strain Y7092 (gift from C. Boone) to generate the query
strain. This query strain was mated to the yeast haploid deletion
collection of non-essential genes (MATa, each gene deleted with
KanMX cassette (confers resistance to G418)). Haploid mutants
harboring the FUS expression plasmid were grown in the presence
of glucose (FUS expression ‘‘off’’) or galactose (FUS expression
‘‘on’’). Following growth at 30uC for 2 d, plates were photo-
graphed and colony sizes measured by ImageJ image analysis
software, based on [104]. The entire screen was repeated three
times and only hits that reproduced all three times were selected
for further validation by random spore analysis on DNA
sequencing of deletion strain bar codes. Deletion strains that grew
poorly on galactose were eliminated based on published data on
deletion strain fitness on galactose as well as in house
measurements using the yeast deletion collection.
FUS Purification
FUS and FUS deletion mutants were expressed and purified
from Escherichia coli as GST-tagged proteins. FUS constructs were
generated in GV13 to yield a TEV protease cleavable GST-FUS
protein, GST-TEV-FUS, and overexpressed in E. coli BL21 DE3
cells (Agilent). Protein was purified over a glutathione-sepharose
column (GE) according to the manufacturer’s instructions.
Proteins were eluted from the glutathione sepharose with
50 mM Tris-HCl pH 8, 200 mM trehalose, and 20 mM gluta-
thione. After purification, proteins were concentrated to 10
mMor
greater using Amicon Ultra-4 centrifugal filter units (10 kDa
molecular weight cut-off; Millipore). Protein was then centrifuged
for 30 min at 16,100 g to remove any aggregated material. After
centrifugation, the protein concentration was determined by
Bradford assay (Bio-Rad) and the proteins were used immediately
for aggregation reactions. GST-TEV-TDP-43 was purified as
described [15].
FUS-RNA binding Assay
RNA-binding assays were performed as described [105]. Briefly,
FUS RNA probe was transcribed by T7 polymerase from DNA
template (59-GTAATACGACTCACTATAGGGGAAAATTAA-
TGTGTGTGTGTGGAAAATT-39) with
32
P-labeled UTP.
Probes were gel-purified and adjusted to 10
4
c.p.m./ml specific
activity. Standard binding reactions were carried out in 10
ml, with
a final concentration of 4 mM MgCl
2
, 25 mM phosphocreatine,
1.25 mM ATP, 1.3% polyvinyl alcohol, 25 ng of yeast tRNA,
0.8 mg of BSA, 1 mM DTT, 0.1
ml Rnasin (Promega, 40 U/ml),
75 mM KCl, 10 mM Tris, pH 7.5, 0.1 mM EDTA, 10% glycerol,
and 0.15
mMto5mM GST-FUS or GST. Binding reactions were
incubated for 20 min at 30uC with
32
P-labeled probe. After
binding, heparin was added to a final concentration of 0.5
mg/ml;
reactions were analyzed on a 4.5% native gel (Acrylamide/Bis
29:1, BioRad).
FUS In Vitro Aggregation Assay
Aggregation was initiated by the addition of TEV protease
(Invitrogen) to GST-TEV-FUS (2.5–5
mM) in assembly buffer
(AB): 100 mM TrisHCl pH 8, 200 mM trehalose, 0.5 mM
EDTA, and 20 mM glutathione. Aggregation reactions were
incubated at 22uC for 0–90 min with or without agitation at
700 rpm in an Eppendorf Thermomixer. No aggregation
occurred unless TEV protease was added to separate GST from
FUS or TDP-43. Turbidity was used to assess aggregation by
measuring absorbance at 395 nm. For sedimentation analysis,
reactions were centrifuged at 16,100 g for 10 min at 25uC.
Supernatant and pellet fractions were then resolved by SDS-
PAGE and stained with Coomassie Brilliant Blue, and the amount
in either fraction determined by densitometry in comparison to
known quantities of FUS. For electron microscopy (EM) of in vitro
aggregation reactions, protein samples (20
ml of a 2.5 mM solution)
were adsorbed onto glow-discharged 300-mesh Formvar/carbon-
coated copper grid (Electron Microscopy Sciences) and stained
with 2% (w/v) aqueous uranyl acetate. Excess liquid was removed,
and grids were allowed to air dry. Samples were viewed using a
JEOL 1010 transmission electron microscope.
Visualizing P-Bodies and Stres s Granules in Yeast
We used fluorescent markers of P-bodies and stress granules and
live cell imaging to monitor stress granule and P-body formation in
yeast, based on standard protocols [63]. First, we transformed
yeast strain BY4741 with pAG423GAL-FUS-YFP. This strain was
then transformed with plasmids encoding P-body markers (Lsm1-
mCherry, LEU2 or Dcp2-RFP, LEU2) or stress granule markers
(Pub1-RFP, URA3 or CFP-Pbp1, URA3) separately. Transfor-
mants were grown overnight to mid-log phase in raffinose-
containing media. To induce expression of FUS-YFP, galactose
was added to 2% and cells were incubated at 30uC for 4 h and
then processed for microscopy. We used a spinning disk confocal
microscope to monitor the YFP, CFP, and RFP signals in live cells.
For each channel, 60 z-sections were acquired at 0.1
mm
increments at 23uC. Figures display the maximum projection of
each channel.
FUS and Modifier Genes Transfection in Mammalian Cells
HEK293T cells were plated in 96-well format and transfected
with FuGene (Roche) according to the manufacturer’s instruc-
tions. 72 h post-transfection, MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) (Sigma) was added to each well
and incubated for 3 h at 37uC. Acidic Isoproponal (40 mM HCl)
was then added to each well to solubilize the blue formazan
crystals. Absorbance of each well was read with a Tecan Safire II
plate reader using 570 nm for absorbance and 630 nm as a
reference wavelength. Absorbance measurements were normalized
to the absorbance of untransfected cells and used to calculate a
percent viability for each condition.
Supporting Information
Figure S1 Prion domain prediction algorithm identifies prion-
like domains in TDP-43 (top) and FUS/TLS (bottom). Note that
Mechanisms of FUS Aggregation and Toxicity
PLoS Biology | www.plosbiology.org 22 April 2011 | Volume 9 | Issue 4 | e1000614
the prion-like domain (PrD) of TDP-43 is located in the C-
terminal region, whereas the PrD of FUS/TLS is in the N-
terminal region. There is an additional peak of PrD character
predicted by the algorithm in FUS/TLS aa 391–407. For
additional details on design and implementation of this prion
domain prediction algorithm, see [33,34].
Found at: doi:10.1371/journal.pbio.1000614.s001 (3.34 MB TIF)
Figure S2 FUS and TDP-43 co-localize in yeast cells. FUS-YFP
and TDP-43-CFP were co-transformed into yeast cells and their
localization visualized by fluorescence microscopy. FUS-YFP and
TDP-43-CFP co-localized to the same subcellular foci (arrows).
Found at: doi:10.1371/journal.pbio.1000614.s002 (1.46 MB TIF)
Figure S3 FUS localizes to the nucleus and cytoplasm when
expressed at lower levels. Yeast strain YEF6030 (YEF473a
NUP57-mCherry-His3), harboring a nuclear envelope marker,
to visualize the nucleus in live cells, was transformed with
416GPD-FUS-YFP. FUS localization in live cells was visualized
using a spinning disc confocal microscope. At this level of
expression, FUS-YFP localized to the nucleus (arrows) and
cytoplasm in a diffuse pattern.
Found at: doi:10.1371/journal.pbio.1000614.s003 (1.98 MB TIF)
Figure S4 FUS truncation proteins localize to the nucleus. DAPI
stained cells confirm nuclear localization of FUS truncation
constructs 1–168aa, 1–269aa, and 1–373aa (also see Figure 3 of
main text).
Found at: doi:10.1371/journal.pbio.1000614.s004 (4.59 MB TIF)
Figure S5 Verifying FUS toxicity modifiers from plasmid
overexpression screen. Spotting assay showing serial dilutions of
yeast cells expressing FUS along with empty vector control, four
enhancers, or six suppressors from the screen.
Found at: doi:10.1371/journal.pbio.1000614.s005 (4.44 MB TIF)
Acknowledgments
We thank Dan Ramos, Pier Hart, and Renske Erion for assistance with the
yeast genetic screens and Sarah Glenn and Kristen Lynch for assistance
with the RNA-binding studies. We thank Jasmine Zhao of the PENN CDB
Microscopy Core for expert assistance with microscopy. We thank Alex
Chavez for providing the CYC1 and NOP1 yeast promoter plasmids and
Jasmine Smith for advice on the SV40 NLS experiments. We thank Roy
Parker for providing some of the yeast stress granule and P-body markers
and Cornelia Kurischko for advice on visualizing stress granules and P-
bodies in yeast. We thank Julien Couthouis and Rose Li for help with some
of the FUS and TDP-43 plasmid constructs. We thank Mark Lemmon and
Morgan DeSantis for comments on the manuscript and helpful suggestions.
We also thank the Petsko and Lindquist laboratories for communicating
their results with us prior to publication.
Author Contributions
The author(s) have made the foll owing declarations about their
contributions: Conceived and designed the experiments: ZS ZD XF
MPH JS ADG. Performed the experiments: ZS ZD XF MPH JS ADG.
Analyzed the data: ZS ZD XF MPH AC JS ADG. Wrote the paper: JS
ADG.
References
1. Rosen D, Siddique T, Patterson D, Figlewicz D, Sapp P, et al. (1993) Mutations
in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic
lateral sclerosis. Nature 362: 59–62.
2. Cleveland DW, Rothstein JD (2001) From Charcot to Lou Gehrig: deciphering
selective motor neuron death in ALS. Nat Rev Neurosci 2: 806–819.
3. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, et al.
(2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and
amyotrophic lateral sclerosis. Science 314: 130–133.
4. Kwong LK, Neumann M, Sampathu DM, Lee VM, Trojanowski JQ (2007)
TDP-43 proteinopathy: the neuropa thology underlying major forms of
sporadic and familial frontotemporal lobar degeneration and motor neuron
disease. Acta Neuropathol 114: 63–70.
5. Chen-Plotkin AS, Lee VM, Trojanowski JQ (2010) TAR DNA-binding protein
43 in neurodegenerative disease. Nat Rev Neurol 6: 211–220.
6. Pesiridis GS, Lee VM, Trojanowski JQ (2009) Mutations in TDP-43 link
glycine-rich domain functions to amyotrophic lateral sclerosis. Hum Mol Genet
18: R156–R162.
7. Gitcho MA, Baloh RH, Chakraverty S, Mayo K, Norton JB, et al. (2008) TDP-
43 A315T mutation in familial motor neuron disease. Ann Neurol 63:
535–538.
8. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, et al. (2008)
TARDBP mutations in individuals with sporadic and familial amyotrophic
lateral sclerosis. Nat Genet 40: 572–574.
9. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, et al. (2008) TDP-43
mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319:
1668–1672.
10. Van Deerlin VM, Leverenz JB, Bekris LM, Bird TD, Yuan W, et al. (2008)
TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropa-
thology: a genetic and histopathological analysis. Lanc et Neurol 7: 409–416.
11. Benajiba L, Le Ber I, Camuzat A, Lacoste M, Thomas-Anterion C, et al. (2009)
TARDBP mutations in motoneuron dis ease with frontotemporal lobar
degeneration. Ann Neurol 65: 470–473.
12. Kovacs GG, Murrell JR, Horvath S, Haraszti L, Majteny i K, et al. (2009)
TARDBP variation associated with frontotemporal dementia, supranuclear
gaze palsy, and chorea. Mov Disord 24: 1843–1847.
13. Gitler AD (2008) Beer and bread to brains and beyond: can yeast cells teach us
about neurodegenerative disease? Neurosignals 16: 52–62.
14. Johnson BS, McCaffery JM, Lindquist S, Gitler AD (2008) A yeast TDP-43
proteinopathy model: exploring the molecular determinant s of TDP-43
aggregation and cellular toxicity. Proc Natl Acad Sci U S A 105: 6439–
6444.
15. Johnson BS, Snead D, Lee JJ, McCaffery JM, Shorter J, et al. (2009) TDP-43 is
intrinsical ly aggregation-pro ne, and amyotroph ic lateral sclero sis-l inked
mutations accelerate aggregation and increase toxicity. J Biol Chem 284:
20329–20339.
16. Elden AC, Kim HJ, Hart MP, Chen-Plotkin AS, Johnson BS, et al. (2010)
Ataxin-2 intermediate-length polyglutamine expansions are associated with
increased risk for ALS. Nature 466: 1069–1075.
17. Kwiatkowski TJ, Jr., Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, et
al. (2009) Mutations in the FUS/TLS gene on chromosome 16 cause familial
amyotrophic lateral sclerosis. Science 323: 1205–1208.
18. Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, et al. (2009)
Mutations in FUS, an RNA processing protein, cause familial amyotrophic
lateral sclerosis type 6. Science 323: 1208–1211.
19. Broustal O, Camuzat A, Guillot-Noel L, Guy N, Millecamps S, et al. (2010)
FUS mutations in frontotemporal lobar degeneration with amyotrophic lateral
sclerosis. J Alzheimers Dis 22: 765–769.
20. Mackenzie IR, Rademakers R, Neumann M (2010) TDP-43 and FUS in
amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neur ol 9:
955–1007.
21. Huang EJ, Zhang J, Geser F, Trojanowski JQ, Strober JB, et al. (2010)
Extensive FUS-immunoreactive pathology in juvenile amyotrophic lateral
sclerosis with basophilic inclusions. Brain Pathol 20: 1069–1076.
22. Munoz DG, Neumann M, Kusaka H, Yokota O, Ishihara K, et al. (2009) FUS
pathology in basophilic inclusion body disease. Acta Neuropathol 118:
617–627.
23. Urwin H, Josephs KA, Rohrer JD, Mackenzie IR, Neumann M, et al. (2010)
FUS pathology defines the majority of tau- and TDP-43-negative frontotem-
poral lobar degeneration. Acta Neuropathol 120: 33–41.
24. Doi H, Koyano S, Suzuki Y, Nukina N, Kuroiwa Y (2010) The RNA-binding
protein FUS/TLS is a common aggregate-interacting protein in polyglutamine
diseases. Neurosci Res 66: 131–133.
25. Woulfe J, Gray DA, Mackenzie IR (2010) FUS-immunoreactive intranuclear
inclusions in neurodegenerative disease. Brain Pathol 20: 589–597.
26. Crozat A, Aman P, Mandahl N, Ron D (1993) Fusion of CHOP to a novel
RNA-binding protein in human myxoid liposarcoma. Nature 363: 640–644.
27. Zinszner H, Sok J, Immanuel D, Yin Y, Ron D (1997) TLS (FUS) binds RNA
in vivo and engages in nucleo-cytoplasmic shuttling. J Cell Sci 110(Pt 15):
1741–1750.
28. Kasyapa CS, Kunapuli P, Cowell JK (2005) Mass spectroscopy identifies the
splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS
as interacting partners of the ZNF198 protein associated with rearrangement in
myeloproliferative disease. Exp Cell Res 309: 78–85.
29. Bertolotti A, Lutz Y, Heard DJ, Chambon P, Tora L (1996) hTAF(II)68, a
novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins
TLS/FUS and EWS is associated with both TFIID and RNA polymerase II.
EMBO J 15: 5022–5031.
30. Fujii R, Okabe S, U rushido T, Inoue K, Yoshimura A, et al. (2005) The RNA
binding protein TLS is translocated to dendritic spines by mGluR5 activation
and regulates spine morphology. Curr Biol 15: 587–593.
Mechanisms of FUS Aggregation and Toxicity
PLoS Biology | www.plosbiology.org 23 April 2011 | Volume 9 | Issue 4 | e1000614
31. Fujii R, Grossenbacher-Zinchuk O, Jamari I, Wang Y, Zinchuk V, et al. (2009)
TLS-GFP cannot rescue mRNP formation near spines and spine phenotype in
TLS-KO. Neuroreport 20: 57–61.
32. Alberti S, Halfmann R, King O, Kapila A, Lindquist S (2009) A systematic
survey identifies prions and illuminate s sequence features of prionogenic
proteins. Cell 137: 146–158.
33. Cushman M, Johnson BS, King OD, Gitler AD, Shorter J (2010) Prion-like
disorders: blurring the divide between transmissibility and infectivity. J Cell Sci
123: 1191–1201.
34. Shorter J, Lindquist S (2005) Prions as adaptive conduits of memory and
inheritance. Nat Rev Genet 6: 435–450.
35. Fuentealba RA, Udan M, Bell S, Wegorzewska I, Shao J, et al. (2010)
Interaction with polyglutamine aggregates reveals a Q/N-rich domain in TDP-
43. J Biol Chem 285: 26304–26314.
36. Udan M, Baloh RH (2011) Implications of the prion-related Q/N domains in
TDP-43 and FUS. Prion :1-5.
37. Lagier-Tourenne C, Polymenidou M, Cleveland DW (2010) TDP-43 and
FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum
Mol Genet 19: R46–R64.
38. Lagier-Tourenne C, Cleveland DW (2009) Rethinking ALS: the FUS about
TDP-43. Cell 136: 1001–1004.
39. Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, et al. (2006) Alpha-
synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s
models. Science 313: 324–328.
40. Gitler AD, Chesi A, Geddie ML, Strathearn KE, Hamamichi S, et al. (2009)
Alpha-synuclein is part of a diverse and highly conserved interaction network
that includes PARK9 and manganese toxicity. Nat Genet 41: 308–315.
41. Scherzinger E, Lurz R, Turmaine M, Mangiarini L, Hollenbach B, et al. (1997)
Hunti ngtin-encoded polyglutamine expansions form amyloid -like protein
aggregates in vitro and in vivo. Cell 90: 549–558.
42. Lashuel HA, Hartley D, Petre BM, Walz T, Lansbury PT, Jr. (2002)
Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature
418: 291.
43. Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, et al. (2009) The
chaperonin TRiC blocks a huntingtin sequence element that promotes the
conformational switch to aggregation. Nat Struct Mol Biol 16: 1279–1285.
44. Giorgini F, Guidetti P, Nguyen Q, Bennett SC, Muchowski PJ (2005) A
genomic screen in yeast implicates kynurenine 3-monooxygenase as a
therapeutic target for Huntington disease. Nat Gen et 37: 526–531.
45. Willingham S, Outeiro TF, DeVit MJ, Lindquist SL, Muchowski PJ (2003)
Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-
synuclein. Science 302: 1769–1772.
46. Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE (2009) Biological and
chemical approaches to diseases of proteostasis deficiency. Annu Rev Biochem
78: 959–991.
47. Gitcho MA, Bigio EH, Mishra M, Johnson N, Weintraub S, et al. (2009)
TARDBP 39-UTR variant in autopsy-confirmed frontotemporal lobar
degeneration with TDP-43 proteinopathy. Acta Neuropathol 118: 633–645.
48. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, et al. (2003) alpha-
Synuclein locus triplication causes Parkinson’s disease. Science 302: 841.
49. Cabrejo L, Guyant-Marechal L, Laquerriere A, Vercelletto M, De la
Fourniere F, et al. (2006) Phenotype associated with APP duplication in five
families. Brain 129: 2966–2976.
50. Huang C, Xia PY, Zhou H (2010) Sustained expression of TDP-43 and FUS in
motor neurons in rodent’s lifetime. Int J Biol Sci 6: 396–406.
51. Bosco DA, Lemay N, Ko HK, Zhou H, Burke C, et al. (2010) Mutant FUS
proteins that cause amyotrop hic lateral sclerosis incorporate into stress
granules. Hum Mol Genet 19: 4160–4175.
52. Dormann D, Rodde R, Edbauer D, Bentmann E, Fischer I, et al. (2010) ALS-
associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated
nuclear import. EMBO J 29: 2841–2857.
53. Gal J, Zhang J, Kwinter DM, Zhai J, Jia H, et al. (2010) Nuclear localization
sequence of FUS and induction of stress granules by ALS mutants. Neurobiol
Aging; E-pub ahead of print. doi:10.1016/j.neurobiolaging.2010.06.010.
54. Kino Y, Washizu C, Aquilanti E, Okuno M, Kurosawa M, et al. (2010)
Intracellular localization and splicing regulation of FUS/TLS are variably
affected by amyotrophic lateral sclerosis-linked mutations. Nucleic Acids Res;
E-pub ahead of print. doi: 10.1093/nar/gkq1162.
55. Ito D, Seki M, Tsunoda Y, Uchiyama H, Suzuki N (2010) Nuclear transport
impairment of amyotrophic lateral sclerosis-linked mutations in FUS/TLS.
Ann Neurol 69: 152–162.
56. Ju S, Tardiff DF, Han H, Divya K, Zhong Q, et al. (2011) A yeast model of
FUS/TLS-dependent cytotoxicity. PLoS Biology;doi: 10.1371/journal.
pbio.1001052.
57. Goldfarb DS, Gariepy J, Schoolnik G, Kornberg RD (1986) Synthetic peptides
as nuclear localization signals. Nature 322: 641–644.
58. Buchan JR, Muhlrad D, Parker R (2008) P bodies promote stress granule
assembly in Saccharomyces cerevisiae. J Cell Biol 183: 441–455.
59. Colombrita C, Zennaro E, Fallini C, Weber M, Som macal A, et al. (2009)
TDP-43 is recruited to stress granules in conditions of oxidative insult.
J Neurochem 111: 1051–1061.
60. Nonhoff U, Ralser M, Welzel F, Piccini I, Balzereit D, et al. (2007) Ataxin-2
interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-
bodies and stress granules. Mol Biol Cell 18: 1385–1396.
61. Ralser M, Nonhoff U, Albrecht M, Lengauer T, Wanker EE, et al. (2005)
Ataxin-2 and huntingtin interact with endophilin-A complexes to function in
plastin-associated pathways. Hum Mol Genet 14: 2893–2909.
62. Freibaum BD, Chitta RK, High AA, Taylor JP (2010) Global analysis of TDP-
43 interacting proteins reveals strong association with RNA splicing and
translation machinery. J Proteome Res 9: 1104–1120.
63. Buchan JR, Nissan T, Parker R (2010) Analyzing P-bodies and stress granules
in Saccharomyces cerevisiae. Methods Enzymol 470: 619–640.
64. Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, et al. (2009) Aberrant
cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad
Sci U S A 106: 7607–7612.
65. Igaz LM, Kwong LK, Chen-Plotkin A, Winton MJ, Unger TL, et al. (2009)
Expression of TDP-43 C-terminal fragments in vitro recapitulates pathological
features of TDP-43 proteinopathies. J Biol Chem 284: 8516–8524.
66. Ayala YM, Zago P, D’Ambrogio A, Xu YF, Petrucelli L, et al. (2008) Structural
determinants of the cellular localization and shuttling of TDP-43. J Cell Sci
121: 3778–3785.
67. Nguyen Ba AN, Pogoutse A, Provart N, Moses AM (2009) NLStradamus: a
simple Hidden Markov Model for nuclear localization signal prediction. BMC
Bioinformatics 10: 202.
68. Dejesus-Hernandez M, Kocerha J, Finch N, Crook R, Baker M, et al. (2010)
De novo truncating FUS gene mutation as a cause of sporadic amyotrophic
lateral sclerosis. Hum Mutat 31: E1377–E1389.
69. Buratti E, Baralle FE (2001) Characterization and functional implications of the
RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of
CFTR exon 9. J Biol Chem 276: 36337–36343.
70. Kim SH, Shanware NP, Bowler MJ, Tibbetts RS (2010) Amyotrophic lateral
sclerosis-associated proteins TDP-43 and FUS/TLS function in a common
biochemical complex to co-regulate HDAC6 mRNA. J Biol Chem 285:
34097–34105.
71. Smit h DB, Johnson KS (1988) Single-step purification of polypeptides
expressed in Escherichia coli as fusions with glutathione S-transferase. Gene
67: 31–40.
72. Forman MS, Trojanowski JQ, Lee VM (2007) TDP-43: a novel neurodegen-
erative proteinopathy. Curr Opin Neurobiol 17: 548–555.
73. Kwong LK, Uryu K, Trojanowski JQ, Lee VM (2008) TDP-43 proteino-
pathies: neurodegenerative protein misfolding diseases without amyloidosis.
Neurosignals 16: 41–51.
74. Frankenfield KN, Powers ET, Kelly JW (2005) Influence of the N-terminal
domain on the aggregation properties of the prion protein. Protein Sci 14:
2154–2166.
75. Hurshman AR, White JT, Powers ET, Kelly JW (2004) Transthyretin
aggregation under partially denaturing conditions is a downhill polymerization.
Biochemistry 43: 7365–7381.
76. Andreu JM, Timasheff SN (1986) The measurement of cooperative protein self-
assembly by turbidity and other techniques. Methods Enzymol 130: 47–59.
77. Baumer D, Hilton D, Paine SM, Turner MR, Lowe J, et al. (2010) Juvenile
ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations.
Neurology 75: 611–618.
78. Ritson GP, Custer SK, Freibaum BD, Guinto JB, Geffel D, et al. (2010) TDP-
43 mediates degeneration in a novel Drosophila model of disease caused by
mutations in VCP/p97. J Neurosci 30: 7729–7739.
79. Kabashi E, Lin L, Tradewell ML, Dion PA, Bercier V, et al. (2010) Gain and
loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor
deficits in vivo. Hum Mol Genet 19: 671–683.
80. Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, et al. (2010) Cytoplasmic
mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation
associated with familial amyotrophic lateral sclerosis. J Neurosci 30: 639–649.
81. Yeger-Lotem E, Riva L, Su LJ, Gitler AD, Cashikar AG, et al. (2009) Bridging
high-throughput genetic and transcriptional data reveals cellular responses to
alpha-synuclein toxicity. Nat Genet 41: 316–323.
82. Hu Y, Rolfs A, Bhullar B, Murthy TV, Zhu C, et al. (2007) Approaching a
complete repository of sequence-verified protein-encoding clones for Saccha-
romyces cerevisiae. Genome Res 17: 536–543.
83. Buchan JR, Parker R (200 9) Eukaryotic stress granules: the ins and outs of
translation. Mol Cell 36: 932–941.
84. Giaever G, Chu AM, Ni L, Connelly C, Riles L, et al. (2002) Functional
profiling of the Saccharomyces cerevisiae genome. Nature 418: 387–391.
85. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, et al. (1996) Life with
6000 genes. Science 274: 546, 563–547.
86. Tong AH, Lesage G, Bader GD, Ding H, Xu H, et al. (2004) Global mapping
of the yeast genetic interaction network. Science 303: 808–813.
87. Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, et al. (2001)
Systematic genetic analysis with ordered arrays of yeast deletion mutants.
Science 294: 2364–2368.
88. Sadlish H, Rampelt H, Shorter J, Wegrzyn RD, Andreasson C, et al. (2008)
Hsp110 chaperones regulate prion formation and propagation in S. cerevisiae
by two dis crete activities. PLoS One 3: e1763. doi:10.1371/journal.pone.
0001763.
89. Gilks N, Kedersha N, Ayodele M, Shen L, Stoecklin G, et al. (2004) Stress
granule assembly is mediated by prion-like aggregation of TIA-1. Mol Biol Cell
15: 5383–5398.
Mechanisms of FUS Aggregation and Toxicity
PLoS Biology | www.plosbiology.org 24 April 2011 | Volume 9 | Issue 4 | e1000614
90. Furukawa Y, Kaneko K, Matsumoto G, Kurosawa M, Nukina N (2009) Cross-
seeding fibrillation of Q/N-rich proteins offers new pathomechanism of
polyglutamine diseases. J Neurosci 29: 5153–5162.
91. Doi H, Okamura K, Bauer PO, Furukawa Y, Shimizu H, et al. (2008) RNA-
binding protein TLS is a major nuclear aggregate-interacting protein in
huntingtin exon 1 with expanded polyglutamine-expressing cells. J Biol Chem
283: 6489–6500.
92. Deng HX, Zhai H, Bigio EH, Y an J, Fecto F, et al. (2010) FUS-
immunoreactive inclusions are a common feature in sporadic and non-
SOD1 familial amyotrophic lateral sclerosis. Ann Neurol 67: 739–748.
93. Ash PE, Zhang YJ, Roberts CM, Saldi T, Hutter H, et al. (2010) Neurotoxic
effects of TDP-43 overexpression in C. elegans. Hum Mol Genet 19:
3206–3218.
94. Liu Y, Eisenberg D (2002) 3D domain swapping: as domains continue to swap.
Protein Sci 11: 1285–1299.
95. Guo Z, Eisenberg D (2006) Runaway domain swapping in amyloid-like fibrils
of T7 endonuclease I. Proc Natl Acad Sci U S A 103: 8042–8047.
96. Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, et al. (2001)
Design of three-dimensional domain-swapped dimers and fibrous oligomers.
Proc Natl Acad Sci U S A 98: 1404–1409.
97. Ling SC, Albuquerque CP, Han JS, Lagier-Tourenne C, Tokunaga S, et al.
(2010) ALS-associated mutations in TDP-43 increase its stability and promote
TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A 107:
13318–13323.
98. Balagopal V, Parker R (2009) Polysomes, P bodies and stress granules: states
and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21: 403–408.
99. Lin CL, Bristol LA, Jin L, Dykes-Hoberg M, Crawford T, et al. (1998)
Aberrant RNA processing in a neurodegenerative disease: the cause for absent
EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron 20:
589–602.
100. Alberti S, Gitler AD, Lindquist S (2007) A suite of Gateway((R)) cloning vectors
for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24:
913–919.
101. Guthrie C, Fink GR (2002) Methods in ezymology: guide to yeast genetics and
molecular and cell biology. Academic Press 169.
102. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast
cells treated with alkali cations. J Bacteriol 153: 163–168.
103. Tong AH, Boone C (2006) Synthetic genetic array analysis in Saccharomyces
cerevisiae. Methods Mol Biol 313: 171–192.
104. Collins SR, Schuldiner M, Krogan NJ, Weissman JS (2006) A strategy for
extracting and analyzing large-scale quantitative epistatic interaction data.
Genome Biol 7: R63.
105. Rothrock CR, House AE, Lynch KW (2005) HnRNP L represses exon splicing
via a regulated exonic splicing silencer. EMBO J 24: 2792–2802.
Mechanisms of FUS Aggregation and Toxicity
PLoS Biology | www.plosbiology.org 25 April 2011 | Volume 9 | Issue 4 | e1000614
    • "Several human proteins have prion-like N/Q-rich domains that have been directly linked to neurodegenerative diseases. Cytoplasmic aggregates of the RNAbinding protein FUS, which contains a Q-rich domain, are implicated in amyotrophic lateral sclerosis, and its aggregation has been re-capitulated in an induced S. cerevisiae proteinopathy [36] . Mutations in two yeastprion-like proteins hnRNPA2B1 and hnRNPA1 initiate neurodegenerative disease in humans through amyloid formation [37]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Prions are transmissible, propagating alternative states of proteins, and are usually made from the fibrillar, beta-sheet-rich assemblies termed amyloid. Prions in the budding yeast Saccharomyces cerevisiae propagate heritable phenotypes, uncover hidden genetic variation, function in large-scale gene regulation, and can act like diseases. Almost all these amyloid prions have asparagine/glutamine-rich (N/Q–rich) domains. Other proteins, that we term here ‘prionogenic amyloid formers’ (PAFs), have been shown to form amyloid in vivo, and to have N/Q-rich domains that can propagate heritable states in yeast cells. Also, there are >200 other S.cerevisiae proteins with prion-like N/Q-rich sequence composition. Furthermore, human proteins with such N/Q-rich composition have been linked to the pathomechanisms of neurodegenerative amyloid diseases. Results Here, we exploit the increasing abundance of complete fungal genomes to examine the ancestry of prions/PAFs and other N/Q-rich proteins across the fungal kingdom. We find distinct evolutionary behavior for Q-rich and N-rich prions/PAFs; those of ancient ancestry (outside the budding yeasts, Saccharomycetes) are Q-rich, whereas N-rich cases arose early in Saccharomycetes evolution. This emergence of N-rich prion/PAFs is linked to a large-scale emergence of N-rich proteins during Saccharomycetes evolution, with Saccharomycetes showing a distinctive trend for population sizes of prion-like proteins that sets them apart from all the other fungi. Conversely, some clades, e.g. Eurotiales, have much fewer N/Q-rich proteins, and in some cases likely lose them en masse, perhaps due to greater amyloid intolerance, although they contain relatively more non-N/Q-rich predicted prions. We find that recent mutational tendencies arising during Saccharomycetes evolution (i.e., increased numbers of N residues and a tendency to form more poly-N tracts), contributed to the expansion/development of the prion phenomenon. Variation in these mutational tendencies in Saccharomycetes is correlated with the population sizes of prion-like proteins, thus implying that selection pressures on N/Q-rich protein sequences against amyloidogenesis are not generally maintained in budding yeasts. Conclusions These results help to delineate further the limits and origins of N/Q-rich prions, and provide insight as a case study of the evolution of compositionally-defined protein domains. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0594-3) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "; Fonte et al., 2008), through knockdown (Fonte et al., 2002; Nollen et al., 2004; Kraemer et al., 2006; Kaltenbach et al., 2007; Hamamichi et al., 2008; Kuwahara et al., 2008; Van Ham et al., 2008; Roodveldt et al., 2009; Wang et al., 2009; Zhang et al., 2010; Calamini et al., 2011; Silva et al., 2011; Brehme et al., 2014; Khabirova et al., 2014; Jimenez-Sanchez et al., 2015), or by introduction of point mutations (Warrick et al., 1999; Chan et al., 2000 Chan et al., , 2002) or deletions (Fernandez-Funez et al., 2000; Krobitsch and Lindquist, 2000; Willingham et al., 2003; Giorgini et al., 2005; Ritson et al., 2010; Butler et al., 2012) in order to explore their role in diseases of protein misfolding. Our survey covers different measures of proteotoxicity, evaluating the enhancement or suppression of various phenotypes as a readout for chaperone functionality, including aggregation (Krobitsch and Lindquist, 2000; Fonte et al., 2002; Nollen et al., 2004; Tam et al., 2006; Wang et al., 2007 Wang et al., , 2009 Hamamichi et al., 2008; Sadlish et al., 2008; Van Ham et al., 2008; Roodveldt et al., 2009; Vos et al., 2010; Zhang et al., 2010; Calamini et al., 2011; Ju et al., 2011; Silva et al., 2011; Sun et al., 2011; Walter et al., 2011; Duennwald et al., 2012; Wolfe et al., 2013), neurodegeneration (Warrick et al., 1999; Chan et al., 2000 Chan et al., , 2002 Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000; Auluck et al., 2002 Auluck et al., , 2005; Shulman and Feany, 2003; Al-Ramahi, 2006; Bilen and Bonini, 2007; Kaltenbach et al., 2007; Hamamichi et al., 2008; Wang et al., 2009; Ritson et al., 2010; Vos et al., 2010; Jimenez-Sanchez et al., 2015), or cellular toxicity (Willingham et al., 2003; Cao et al., 2005; Giorgini et al., 2005; Cooper et al., 2006; Kraemer et al., 2006; Kuwahara et al., 2008; Liang et al., 2008; Yeger-Lotem et al., 2009; Elden et al., 2010; Vos et al., 2010; Calamini et al., 2011; Ju et al., 2011; Sun et al., 2011; Butler et al., 2012; Wolfe et al., 2013 Wolfe et al., , 2014 Brehme et al., 2014; Khabirova et al., 2014; Kim et al., 2014). Based on this collection of studies, we first review findings based on the use of a defined single candidate gene to demonstrate a role for each of the major chaperome families in modifying proteotoxicity. "
    [Show abstract] [Hide abstract] ABSTRACT: Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases.
    Full-text · Article · Aug 2016
    • "While the prion-like domains of TDP-43 and FUS have been incriminated for their aggregation propensity, other protein regions seem to be contributing to this behavior. The FUS prion-like domain alone is insufficient to cause aggregation, but requires the RNA-binding motif and a glycine-rich RGG domain (Sun et al. 2011). Intriguingly, this RGG domain contains a short low complexity region (amino acids 391–407), which might allow the protein to self-aggregate using its two prion-like domains (Gitler and Shorter 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Frontotemporal dementia is a devastating neurodegenerative disease causing stark alterations in personality and language. Characterized by severe atrophy of the frontal and temporal brain lobes, frontotemporal dementia (FTD) shows extreme heterogeneity in clinical presentation, genetic causes, and pathological findings. Like most neurodegenerative diseases, the initial symptoms of FTD are subtle, but increase in severity over time, as the disease progresses. Clinical progression is paralleled by exacerbation of pathological findings and the involvement of broader brain regions, which currently lack mechanistic explanation. Yet, a flurry of studies indicate that protein aggregates accumulating in neurodegenerative diseases can act as propagating entities, amplifying their pathogenic conformation, in a way similar to infectious prions. In this prion-centric view, FTD can be divided into three subtypes, TDP-43 or FUS proteinopathy and tauopathy. Here, we review the current evidence that FTD-linked pathology propagates in a prion-like manner and discuss the implications of these findings for disease progression and heterogeneity. Frontotemporal dementia (FTD) is a progressive neurodegenerative disease causing severe personality dysfunctions, characterized by profound heterogeneity. Accumulation of tau, TDP-43 or FUS cytoplasmic aggregates characterize molecularly distinct and non-overlapping FTD subtypes. Here, we discuss the current evidence suggesting that prion-like propagation and cell-to-cell spread of each of these cytoplasmic aggregates may underlie disease progression and heterogeneity. This article is part of the Frontotemporal Dementia special issue.
    Full-text · Article · Aug 2016
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