The toxicity of an "artificial" amyloid is related to how it interacts with membranes.
ABSTRACT Despite intensive research into how amyloid structures can impair cellular viability, the molecular nature of these toxic species and the cellular mechanisms involved are not clearly defined and may differ from one disease to another. We systematically analyzed, in Saccharomyces cerevisiae, genes that increase the toxicity of an amyloid (M8), previously selected in yeast on the sole basis of its cellular toxicity (and consequently qualified as "artificial"). This genomic screening identified the Vps-C HOPS (homotypic vacuole fusion and protein sorting) complex as a key-player in amyloid toxicity. This finding led us to analyze further the phenotype induced by M8 expression. M8-expressing cells displayed an identical phenotype to vps mutants in terms of endocytosis, vacuolar morphology and salt sensitivity. The direct and specific interaction between M8 and lipids reinforces the role of membrane formation in toxicity due to M8. Together these findings suggest a model in which amyloid toxicity results from membrane fission.
- SourceAvailable from: Karine V.J. Berthelot[show abstract] [hide abstract]
ABSTRACT: REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.PLoS ONE 01/2012; 7(10):e48065. · 3.73 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Many studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. Previously, we generated a yeast toxic amyloid mutant (M8) from the harmless amyloid protein by changing a few residues of the Prion Forming Domain of HET-s (PFD HET-s(218-289)) and clearly demonstrated the complete different behaviors of the non-toxic Wild Type (WT) and toxic amyloid (called M8) in terms of fiber morphology, aggregation kinetics and secondary structure. In this study, we compared the interaction of both proteins (WT and M8) with membrane models, as liposomes or supported bilayers. We first demonstrated that the toxic protein (M8) induces a significant leakage of liposomes formed with negatively charged lipids and promotes the formation of microdomains inside the lipid bilayer (as potential "amyloid raft"), whereas the non-toxic amyloid (WT) only binds to the membrane without further perturbations. The secondary structure of both amyloids interacting with membrane is preserved, but the anti-symmetric PO(2)(-) vibration is strongly shifted in the presence of M8. Secondly, we established that the presence of membrane models catalyzes the amyloidogenesis of both proteins. Cryo-TEM (cryo-transmission electron microscopy) images show the formation of long HET-s fibers attached to liposomes, whereas a large aggregation of the toxic M8 seems to promote a membrane disruption. This study allows us to conclude that the toxicity of the M8 mutant could be due to its high propensity to interact and disrupt lipid membranes.Biochimica et Biophysica Acta 04/2012; 1818(9):2325-34. · 4.66 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a "raft-like" mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.Biochimie 07/2012; · 3.14 Impact Factor
www.landesbioscience.com Prion 283
Prion 4:4, 283-291; October/November/December 2010; © 2010 Landes Bioscience
The toxicity of an “artificial” amyloid is related
to how it interacts with membranes
*Correspondence to: Christophe Cullin; Email: Christophe.Cullin@ibgc.u-bordeaux2.fr
Submitted: 06/08/10; Accepted: 07/23/10
Previously published online: www.landesbioscience.com/journals/prion/article/13126
Amyloid proteins form a broad group of proteins that share the
ability to display a particular quaternary structure: amyloid
fibrils. Although unrelated in terms of their primary sequence
and function, these proteins share common structural charac-
teristics, such as fibrillar polypeptide aggregates with cross-beta
protein conformations. Most studies have converged on amyloid
proteins related to various diseases.1 Indeed, the pathological pro-
cesses associated with Parkinson, Alzheimer or prion diseases are
due to the aggregation of α-synuclein (α-syn), Amyloid-ß (Aß)
or Prp proteins, respectively. However, amyloids are also used for
normal cellular functions.2 This is the case in unicellular cells
(in E. coli and S. cerevisiae) in which amyloid proteins allow the
fixation of cells onto a glass surface and the formation of bio-
films, but amyloid proteins may also form prions that can help
the cells to survive in particular conditions.3-6 This is also the
case in metazoans with Pmel17, which is involved in mammalian
skin pigmentation.7 Also, several endocrine hormones appear to
be stored in secretory granules in an amyloid-like state.8
Interestingly, both toxic and non-toxic amyloids share com-
mon or closely related oligomeric species. These oligomeric
species are produced early during polymerization kinetics and
are recognized by the same antibodies. These antibodies are
directed against artificially made oligomers, synthesized by cou-
pling an Aß peptide to the surface of colloidal gold beads via its
C-terminus. The assembly of toxic and immunoreactive species
Despite intensive research into how amyloid structures can impair cellular viability, the molecular nature of these toxic
species and the cellular mechanisms involved are not clearly defined and may differ from one disease to another. We
systematically analyzed, in Saccharomyces cerevisiae, genes that increase the toxicity of an amyloid (M8), previously
selected in yeast on the sole basis of its cellular toxicity (and consequently qualified as “artificial”). This genomic
screening identified the Vps-C HOPS (homotypic vacuole fusion and protein sorting) complex as a key-player in amyloid
toxicity. This finding led us to analyze further the phenotype induced by M8 expression. M8-expressing cells displayed
an identical phenotype to vps mutants in terms of endocytosis, vacuolar morphology and salt sensitivity. The direct and
specific interaction between M8 and lipids reinforces the role of membrane formation in toxicity due to M8. Together
these findings suggest a model in which amyloid toxicity results from membrane fission.
Julien Couthouis,† Christelle Marchal,† Fabien D’Angelo, Karine Berthelot and Christophe Cullin*
IBGC; UMR 5095; CNRS; Université Bordeaux 2 Victor Segalen; Bordeaux, France
†These authors contributed equally to this work.
Key words: aggregates, amyloid, yeast, euroscarf
reaches a maximal level at the same time during polymerization,
leading to the conclusion that these oligomeric species affect cel-
lular viability.9,10 The link between the formation of toxic spe-
cies and its structural requirements remains poorly understood.
This is due in part to the experimental approaches used (typi-
cally based on adding large concentrations of proteins to cell cul-
tures), as they do not represent the complexity of amyloid cellular
toxicity accurately. We recently set-up a genetic screen based on
generating toxic amyloids from an originally harmless amyloid.11
Our model used the yeast S. cerevisiae to monitor the cellular
toxicity of amyloids. Although the budding yeast does not allow
modeling of complex neuronal diseases, such as Parkinson’s or
Alzheimer’s, it does however pinpoint the role of factors involved
in this process. For instance, eight of nine toxicity modifiers iden-
tified in yeast had similar effects on α-synuclein toxicity in yeast
and in neuronal systems.12 This model may therefore represent an
attractive alternative model for studying amyloid toxicity.
The harmless amyloid peptide used in this study was the
prion domain of the Het-s protein from P. anserina. The more
toxic mutant (Mutant 8, M8) differs from the wild-type by ten
mutations. In vivo, this mutant forms smaller dotted aggregates,
which differ from the large annular wild-type (WT) aggregates.
We then biochemically and biophysically characterized the struc-
tural characteristics and differences in this toxic M8 mutant.13
In vitro, the fibers formed by WT and M8 amyloids differ sig-
nificantly. WT amyloids exhibit typical μm-long fibers when
observed through transmission electronic microscopy, whereas
284 Prion Volume 4 Issue 4
Remaining non-transformable strains (auxotrophic or unable to
grow on our minimal media) were not analyzed in this study. These
remaining 113 strains are listed in Supplementary Table 2.
Strains able to grow on both dextrose or galactose media. These
strains were analyzed and screened for M8 toxicity enhancers.
At the end of this selection process, 72 strong toxicity enhancers
were found for M8. One drawback to the yeast deletion library
is that some strains potentially share an additional unexpected
mutation.18 As the same putative mutation cannot be present in a
strain bearing the same KO but genetically independent from the
previous library, we have checked for the toxicity phenotype in
the opposite mating type (Mat a) strains obtained independently.
We also tested for toxicity specificity using WT Het-s amyloid
and finally isolated only 46 of the selected strains as strong M8
toxicity enhancers (Suppl. Table 3). Among these 46 strains,
three also increase WT toxicity. Forty-four of these 46 mutants
were completely different from those previously identified as
α-synuclein and poly-glutamine toxicity enhancers,15 suggesting
alternative toxicity pathways for M8 toxicity. We further investi-
gated the mechanism underlying M8 toxicity by classifying the
identified genes into functional categories.
Gene ontology classification highlights the role of the
vacuole. Identified genes were classified using the FunSpec algo-
rithm,19 located at the Munich Information Center for Protein
Sequences (MIPS) comprehensive yeast genome database and
the Gene Ontology (GO) database. These classification methods
helped us to identify possible clusters of genes involved in the
same mechanism. In our case, it highlighted a group of genes
involved in cellular traffic, which were strongly linked to vacu-
olar pathways (Table 1). Thirty percent of proteins encoded by
the selected genes were localized in membranes, mitochondria
and vacuoles, and were functionally involved in membrane orga-
nization and vesicle-mediated transport. Among these identified
genes, three genes (PEP3, PEP5 and VPS16) belong to the class C
VPS family. The Vps-C complexes contain the CORVET (class
C core vacuole/endosome tethering) and HOPS (homotypic
vacuole fusion and protein sorting) complexes. Both are found
in endosomes and vacuoles, in which they promote membrane
fusion. These complexes are built on a common Vps-C core
complex formed by the products of four genes (PEP3 (VPS18),
VPS16, PEP5 (VPS11 or END1) and VPS33 (SLP1)). The YPT7
gene is also involved in vacuolar function, but is not part of the
Vps-C core complex. We thus characterized, in more detail, the
reasons for which M8 expression is more deleterious in these
M8 selectively inhibits the growth of class C vps mutants.
Defective growth, scored by serial dilution, did not allow the
identification of whether the changes occurred during the lag,
exponential or stationary phases. We analyzed the toxic effects
of M8 in class C vps-deleted strains more precisely; thus, we
checked their growth against similar strains expressing WT amy-
loid or GFP alone (Fig. 1). BY4742 cells expressing WT or GFP
reached the stationary phase 48 h after induction. Cells express-
ing M8 grew more slowly, but reached the same density after 96 h
of induction (A). The stationary phase was reached later in pep3,
pep5 and vps16 class C vps mutants (D–F) expressing M8, and
M8 amyloids assemble into 80 nm-long filaments. Wide Angle
X-ray Scattering (WAXS) and ATR-FTIR spectroscopy con-
firmed that M8 is also an amyloid, but with a totally different
secondary structure based on antiparallel b-sheets. However,
one major question continues to persist: what makes an amyloid
Here, we have used a library of yeast gene deletion strains,
known as the Euroscarf Library.14 This yeast KO library has
been successfully used to identify genes modulating cellular tox-
icity of Huntingtin with poly-glutamine (poly-Q) expansions
and α-synuclein.15 This collection contains 4,850 viable mutant
haploid strains, each lacking a single gene. These strains were
manually and individually transformed with constructs allowing
conditional expression of the previously isolated toxic mutant M8.
From these strains, we isolated 46 gene deletions that enhance
M8 toxicity. Interestingly, the isolated genes were different from
those previously identified as modulators of α-synuclein or poly-
Q expressions. This set of isolated genes clearly highlights the
role of traffic vesicles as a key factor in cellular toxicity. High M8
affinity for DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocho-
line) and changes in cellular traffic due to its expression led us to
suggest a model in which M8 toxicity occurs due to a greater pro-
pensity for it to be inserted into lipid layers, leading to vesicular
trafficking “poisoning,” through a vesicular fission mechanism.
Yeast knockout library screening reveals few toxicity
enhancers. Selecting deleted strains in which M8 toxicity was
enhanced involved three steps. First, all strains from an ordered
and frozen library were transformed using the microtiter plate
transformation method.16 This method was slightly tailored to
the materials in our lab (see Materials and Methods).
Isolates of interest were those in which growth defects were
specifically correlated with M8 expression. These clones were
separated from false positive strains in which the growth defect
may be due to the deletion itself or the transformation process.
Thus, after the transformation step, we plated yeasts on dex-
trose medium for plasmid selection and non-selective galactose
medium. The dextrose medium ensured the selection of trans-
formed strains and prevented M8 expression. By contrast, the
cell suspension grown on non-selective galactose medium was
mostly populated with untransformed cells not expressing M8.
The comparison between these two conditions allowed us to cat-
egorize the Euroscarf strains into three groups (Fig. S1).
Strains unable to grow on galactose medium but forming colo-
nies on the dextrose plate. These strains were unable to grow on
medium in which galactose was the sole carbon source. These
strains were classified as false positives (as growth inhibition was
not due to M8 but due to their incapacity to grow under the
experimental conditions outlined). The 271 (Gal-) strains identi-
fied are listed in Supplementary Table 1.
Strains unable to grow on both dextrose or galactose media. These
were either auxotrophic strains or strains for which the microtiter
transformation protocol was ineffective. These strains were then
individually re-transformed via a standard acetate lithium protocol.17
www.landesbioscience.com Prion 285
Sensitivity to metals and salts. We first explored the sensitiv-
ity of BY4742 cells expressing GFP, WT or M8 to LiCl, CaCl2,
ZnCl2, MgCl2, MnCl2 and citric acid (Fig. 2). Growth was slower
in all strains when plated on medium containing these molecules,
but the presence of M8 did indeed increase the sensitivity of the
yeast to these salts and metals. As control, we have verified that
the three strains pep3Δ, pep5Δ and vps16Δ expressing GFP or
WT are sensitive to these molecules (Fig. 2B). It is difficult to
measure the extent of salt sensitivity of M8 expression in BY4742
vs. deleted strains since without any chemical compounds these
strains do not grew in the same way. However, the sensitivity of
strains expressing M8 or bearing deletions appears to be roughly
Vacuolar morphology. We then analyzed vacuolar morphol-
ogy by staining cells with lipophilic dye FM4-64. This vital
fluorescent dye initially stains the plasma membrane and is then
internalized and delivered to the vacuolar membrane.24 Cells
were incubated with FM4-64 for 10 min, washed, and subse-
quently incubated with FM4-64 for up to 45 min at room tem-
perature. The presence of GFP or WT did not disturb vacuolar
morphology (Fig. 3A and B). By contrast, several vesicles were
placed side by side in M8 cells, suggesting that M8 probably trig-
gers vacuole fragmentation (Fig. 3C).
Lipid binding. As amyloid toxicity is often related to the
ability of these proteins to interact directly with membrane25 we
then analyzed the ability of M8 to directly bind to phosphati-
dylcholine (PC). The purified M8 peptide binds to DMPC (a
representative component of PC in yeast membranes)26 (Fig. 4).
the plateau was lower, indicating a higher mortality for the yeast
Class C vps-deleted strains exhibited slower growth than wild
type strains. Thus, their behavior in the presence of M8 may
have been due to the combination of two other growth-slowing
factors. We then investigated how other KO strains are affected
by WT, M8 or GFP. These other KO strains (cpr7Δ and adk1Δ
strains20,21) were selected for their decreased rate of growth during
the exponential phase. These strains have a slower growth, but
reached the same plateau (stationary phase) whichever protein
was expressed (WT, M8 or GFP) (Fig. 1B and C). Thus, toxicity
enhancement observed in class C vps-deleted strains induced by
M8 is specifically related to the production of M8, and does not
result from other unconnected effects that slow growth. One triv-
ial explanation for increasing M8 toxicity may be the capacity for
vacuolar mutants to stabilize M8, thus leading to increasing its
concentration up to a deleterious threshold level. However, west-
ern-blot analysis of M8 expressed in PEP3Δ, PEP5Δ or VPS16Δ
does not sustain this hypothesis (Suppl. Fig 2). We then studied
how M8 affects vacuolar protein sorting, as mutations in this cel-
lular process increase its toxicity.
M8 interacts with lipids and affects cellular trafficking. We
investigated whether the presence of M8 results in a phenotype
mimicking pep3Δ, pep5Δ and vps16Δ phenotypes. These mutants
exhibit sensitivity to several metals or salts,22 present some modi-
fications of their vacuolar morphology, and display some defects
in vesicular traffic via an alteration of endocytosis and cargo
delivery to the vacuole.23
Table 1. MIPS and GO classification of verified M8 toxicity enhancers
MIPS functional classificationp-valueGenes in category from clusterkf
Vesicular transport 1.50E-03PEP3 YPT7 PEP5 VPS16472
Vacuole or lysosome3.43E-03 PEP3 PEP5 VPS163 44
CHC1 PEP3 YPT7 PEP5 VPS16
Genes in category from cluster
Other vacuolar mutants1.99E-05PEP8 PEP3 YPT7 PEP5 VPS165 49
Sporulation efficiency1.01E-03UME6 PEP3 PEP5 IRA2 VPS165 112
GO biological process
MDM10 TPD3 TPS2 CHC1 UBI4 PEP3 END3 VPS16
Genes in category from cluster
Golgi to endosome transport 1.09E-04PEP3 PEP5 VPS163 14
Vacuole fusion, non-autophagic 1.57E-04PEP3 YPT7 PEP5 VPS164 40
Mitotic cell cycle spindle assembly checkpoint 7.35E-04 TPD3 BUB1 BUB33 26
Late endosome to vacuole transport1.24E-03PEP3 PEP5 VPS163 31
Negative regulation of protein metabolic process1.36E-03TWF1 BUB1 BUB33 32
Vacuolar transport2.25E-03 PEP8 PEP3 PEP5338
Negative regulation of transcription, mitotic 6.97E-03UME611
Positive regulation of meiosis6.97E-03UME611
Regulation of membrane potential6.97E-03PMP311
Sequestering of actin monomers6.97E-03TWF111
Vesicle docking during exocytosis9.17E-03PEP3 PEP52 21
Identified genes were clustered according to their function, phenotype or implied biological process. k is the number of genes from the input cluster
in given category and f the total number of genes in given category.
286 Prion Volume 4 Issue 4
Figure 1. Viability assays for WT and KO strains. Growth of BY4742 or mutant cells expressing GFP (▲), WT (◆) or M8 (■). Cells were grown overnight
on dextrose medium supplemented with casaminoacids (0.67%). When cells reached the exponential phase, they were transferred into galactose
medium supplemented with casaminoacids (0.05 OD600 nm/ml). Cell concentrations were evaluated by OD600 nm at various times after the induction over
a period of 120 h.
Figure 2. Metal and salt sensitivity spotting assay. Ten-fold dilutions of exponentially growing cultures of BY4742 cells expressing GFP, WT or M8 were
spotted onto SD agar supplemented with casaminoacids (0.67%) and containing 8 mM MnCl2, 100 mM MgCl2, 25 mM LiCl, 100 mM CaCl2 or 10 mM
citric acid (A). pep3Δ, pep5Δ and vps16Δ expressing GFP or WT were tested in the same conditions (B). The cells were incubated at 30°C for 4 days.
www.landesbioscience.com Prion 287
amount of uracil or 5-FU into the cell, leading to its death.27 We
performed spotting assays by plating serial dilutions of BY4742
cells expressing GFP, WT and M8 onto two types of media:
containing 5-FU or not (Fig. 5A). After several days, whereas
M8 expressing cells no longer display any toxicity phenotype as
they reached the stationary plateau on a plate without 5-FU, cells
producing M8 and grown on a 5-FU containing medium still
display a toxicity phenotype due to their increased sensitivity to
5-FU, indicating a disturbance of endocytosis.
We then investigated the traffic between the Golgi and vacu-
ole. Resident proteins of the vacuole are synthesized and translo-
cated from the cytosol into the lumen of the ER or inserted into
Under similar experimental conditions, WT was not found to be
associated with this phospsholipid, as it was not detected on the
filter with the fatty acid. This absence of detection was indepen-
dent of the method used (direct observation with Ponceau red or
immunodection). M8 binds to phospholipids, and thus we also
investigated its ability to modify membrane traffic.
Vesicular trafficking. We monitored endocytic traffic by
measuring the transport of 5-fluoro-uracil (5-FU), a toxic analog
of uracil that is imported by the permease Fur4p. In wild-type
cells, Fur4p is continually removed from the plasma membrane
by endocytosis. If endocytosis slows down or is blocked, Fur4p
accumulates at the plasma membrane, and allows a significant
Figure 3. Vacuolar morphology observation by microscopy. Exponentially growing cultures of BY4742 cells expressing GFP, WT or M8 were incubated
for 15 min on ice with the fluorescent marker FM 4–64 (20 μM). Then, the cells were washed twice and were incubated for 45 min at 30°C. Cells were
mounted in medium and observed with a DMRB microscope with a 100X HCX PL fluotar objective.
288 Prion Volume 4 Issue 4
the ER, where it undergoes signal peptide cleavage and N-linked
core-glycosylation generating precursor 1CPY (p1:67 kDa). In
the Golgi, CPY undergoes additional glycosylation, generating
p2CPY (69 kDa). CPY is finally transported via late endosomes
to the vacuole, where it is processed to give the 61 kDa mature
species (mCPY).29 As expected, vps mutants accumulate p2 CPY
(Fig. 5B), and this species is secreted into the medium where
it is detected by colony immunoblotting (data not shown). By
contrast, we detected no defect in CPY processing in M8 cells:
p2CPY does not accumulate and is not secreted in the medium
(Fig. 5B). Similar results were obtained during studies of ALP
processing (data not shown). Thus, M8 does not prevent the sort-
ing of proteins and their transfer to the vacuole.
We have previously carried out a structure-toxicity analysis of
an amyloid protein that is well characterized at the biochemi-
cal level.30 We aimed to isolate mutants on the basis of their
toxicity and to compare their properties with those of the WT
amyloid. This unbiased screen isolated a mutant containing 10
single mutations. Indeed, this mutant M8 is quite different from
the WT, both in vivo and in vitro. M8 forms smaller aggregates
and displays rapid polymerization, involving highly structured
intermediates.31 Biochemical characterization is only part of the
answer, as information on toxicity results from both biochemical
and cellular data. Here, we used a whole-genome approach to
determine the mutations that modulate in trans the toxicity due
to M8 expression. The yeast deletion library screening procedure
isolated 46 KO mutants, of which two were previously shown to
be involved in amyloid toxicity. One of these mutants (yol049WΔ)
displays a partial loss of viability on the overproduction of exon
1 of human HD protein.15 The corresponding gene encodes for
a glutathione synthetase carrying out glutathione synthesis from
γ-glutamylcysteine and glycine.32 The corresponding GSH-
deficient mutant dies rapidly if the cells are directly exposed to
lethal temperatures.33 Thus, this gene may have a general role
in protection and may not be specific to amyloid toxicity. The
yol108CΔ strain has already being identified as a modulator of
α-synuclein toxicity.15 This gene encodes a transcriptional activa-
tor of the ENO1 gene,34 an enolase required for vacuole fusion
and protein transport to the vacuole.35 Interestingly, oxidative
inactivation of human ENO1 (the corresponding ortholog) may
lead to the development of Alzheimer disease.36 The link with the
vacuole was reinforced by the presence of 3 of the 4 members of
the Vps-C core complex. As previously described, knocking out
these genes leads to the complete absence of the vacuole.23 This
absence may be in part responsible for an M8 toxicity increase,
through the relocation of the toxic amyloid to another compart-
ment in which it could have a deleterious effect on the cell. This
suggestion is, however, unlikely, as M8 does not appear to be
specifically localized to the vacuole.11 Moreover, the identification
of YPT7 (a Rab-GTPase involved in binding the Vps-C HOPS
complex to the vacuole membrane37) as a modulator of M8 toxic-
ity argues for a role of the Vps-C complex that is independent to
the presence of the vacuole. The vacuole is highly fragmented in
the ER membrane. They then move to the Golgi. Two pathways
mediate transport between the late Golgi and the vacuole: vacu-
olar hydrolases, such as carboxypeptidase Y (CPY), are delivered
to the vacuole via an endosome-like compartment, but alka-
line phosphatases (ALP) bypass the endosome and are directly
delivered to the vacuole.28 The intracellular trafficking of pro-
teins along these pathways is accompanied by post-translational
modification, glycosylation and proteolytic processing.29 CPY is
synthesized as a precursor that is translocated into the lumen of
Figure 4. M8 interacts specifically with lipids in vitro. M8 interacts
with DMPC blotted onto a PVDF membrane. M8 is detected directly by
Ponceau red and indirectly by an anti-histidine antibody.
Figure 5. Traffic monitoring. (A) Ten-fold dilutions of exponentially
growing cultures of BY4742 cells expressing GFP, WT or M8 were spot-
ted onto SG agar with or without 5 μM 5-FU. Petri dishes were observed
after 10 days at 30°C to allow the observation of M8 growth. (B) Expo-
nentially growing cultures of BY4742 or mutant cells expressing GFP or
M8 were used to prepare total cell extracts. The samples were run on a
12% SDS-PAGE gel, transferred onto a nitrocellulose membrane and ex-
posed to anti-CPY antibodies. m, mature protein; p, precursor protein.
www.landesbioscience.com Prion 289
pep3Δ (MATα, his3Δ1, leu2Δ0,ura3Δ0, YLR148W::kanMX4),
pep5Δ (MATα, his3Δ1,leu2Δ0, ura3Δ0, YMR231W::kanMX4)
vps16Δ (MATα,his3Δ1, leu2Δ0, ura3Δ0, YPL045W::kanMX4).
As specified, yeasts were grown in SD medium (0.67% yeast
nitrogen base, 2% dextrose) or SG medium (0.67% yeast nitro-
gen base, 2% galactose) supplemented with 20 mg/L histidine
(H), 20 mg/L lysine (K) and 60 mg/L leucine (L) or 0.67% casa-
minoacids. We used multicopy (2 μ) yeast expression plasmids
with the URA3 selectable marker in this study: they include
pYeYGFP2U (GFP), pYecHetsYGFP2U (WT), pYecHetsm8YG-
FP2U (M8).11 The fusion of the HET-s(PrD) and yeast opti-
mized GFP42 is expressed under control of a GAL10 promoter in a
pYeHFN2U.43 pYEF2mtRFP allows expression of mitochondrial
RFP. It is derived from pYEF1mtRFP,44 which was constructed
using PYX-mtGFP.45 The selectable marker was changed from
URA3 (pYEF1mtRFP) to LEU2 (pYEF2mtRFP).
Microtiter plate transformations. We used the microtiter
plate transformation method16 with a few modifications. We used
6 μg of carrier DNA (Ozyme) per well. To lower the cross-con-
tamination risks during supernatant elimination, we also covered
the sink used to recover the supernatant with paper. We also used
a 48-well replicator to plate solutions onto standard Petri dishes.
Moreover, we used pipette tips specific for viscous solutions, i.e.,
with a larger opening (Dutcher), particularly for solutions with
Spotting assay. All spotting assays were performed under
identical conditions. Tenfold serial dilutions starting with an
equal number of cells (1OD; l = 600 nm) were prepared in ster-
ile water. Three independent fresh transformant samples were
pooled for the spotting assays. Five-microliter drops were then
plated onto SD or SG medium.
Selecting clones of interest. After a one-night recovery cul-
ture on rich dextrose solid medium (YPD), frozen yeasts from
the Euroscarf library were screened using the Gietz microtiter
plate transformation protocol. A two-step selection was then
ypt7Δ cells, and we found a similar phenotype if M8
was expressed in yeast cells. This phenotype indicates
that M8 has the capacity to interfere in the equilibrium
between fusion and fission of vacuolar vesicles, but
does not elicit its toxic effect through vacuolar function
M8 expression failed to block the traffic between the
Golgi apparatus and the vacuole, including cargo deliv-
ery to the vacuole, as carboxypeptidase Y and alkaline
phosphatase are efficiently processed in the presence
of M8. Interestingly, M8 impaired but did not sup-
press endocytosis, as FM4-64 was still internalized in
yeast cells. Thus, the presence of M8 in the cell trig-
gers some of the phenotypes observed in class C vps-
deleted strains (increased sensitivity to metals or salts
and impairment of endocytic traffic), but this effect was
significantly attenuated. The synergetic effects between
M8 and class-C vps mutants raise the possibility, in
addition to their involvement in endosome-to-vacuole
transport, that M8 and the class-C VPS complex play
additional roles at other transport steps. This sugges-
tion is consistent with the role of the class-C VPS complex in the
processes of membrane docking and fusion at both the Golgi-
to-endosome and endosome-to-vacuole stages of transport.38 M8
affinity for lipids may lead directly to vesicle fission. In our model
(Fig. 6), M8 antagonizes the fusion function of the Vps-C HOPS
complex. In the absence of this complex, small vesicle formation
is favored, leading to significantly greater growth impairment.
The target vesicles may be vacuolar vesicles or other endocyto-
sis vesicles. Mitochondria were excluded, as M8 did not affect
mitochondrial networks and its toxicity remained the same in
rho° cells (Suppl. Fig. 1). This mechanism echoes the recent find-
ing on α-synuclein toxicity in which a genome-wide analysis has
linked its toxicity in yeast to endocytosis of the protein and vacu-
olar fusion defects.39 It may also be correlated to the disruption of
endocytosis which was earlier identified as one of the causes for
cellular toxicity induced by aggregated poly-glutamines.40 Our
model is also strengthened by a recent study based on in vitro
experiments linking disruption of liposome membranes and loss
of cell viability to amyloid fibril length.41 Unusual M8 amyloid
characteristics (short fibrils of 80 nm), together with the presence
of oligomeric intermediates, are consistent with a model in which
toxicity results from membrane disruption. Our findings, based
on an unbiased screen to isolate a new and artificial toxic amy-
loid and to search for genes modulating its toxicity through trans
interactions, showed that membrane fusion/fission is an essential
phenomenon in amyloid toxicity and offers a powerful model for
studying this phenomenon at the molecular level.
Materials and Methods
Yeast strains, media and plasmids. Yeast strains used are isogenic
to BY4742 strain (MATα, his3D1, leu2D0, ura3D0). All deletion
strains were from the Euroscarf yeast deletion library :
adk1Δ (MATα, his3Δ1, leu2Δ0,ura3Δ0, YDR226W::kanMX4)
cpr7Δ (MATα, his3Δ1, leu2Δ0,ura3Δ0, YJR032W::kanMX4)
Figure 6. M8 affects vesicle trafficking. The class C HOPS complex promotes small
vesicles, like early endosomes, fusion to the vacuole. M8 may prevent this fusion
by coating the vacuole with an hydrophobic layer of proteins. Both M8 expression
and the absence of the class C HOPS complex will affects vesicle formation
leading to an accumulation of small vesicles and to a significantly greater growth
impairment. Target vesicles may be vacuolar vesicles or may be other endocytosis
290 Prion Volume 4 Issue 4
and separated on a 12% SDS-PAGE. Proteins were electrically
transferred onto nitrocellulose membranes (Optitran BA-S83,
Schleicher & Schuell) in the presence of transfer buffer (39 mM
Glycine, 48 mM Tris-base, 2% EtOH and 0.037% SDS) and
were probed with monoclonal 0.5 μg/ml anti-CPY antibodies
(Molecular Probes). Peroxidase-conjugated anti-mouse anti-
bodies (Sigma) were used as secondary antibodies. Binding was
detected with the SuperSignal reagent (Pierce) and the VersaDoc
Imaging system (BioRad).
Protein/lipid interactions in vitro. WT and M8 histi-
dine-tagged proteins were produced as described previously.13
Proteins in urea were desalted in 10 mM HCl pH 2.0. To start
polymerisation, native proteins were prepared at concentra-
tions of 20 μM in sterile water and pH was risen to 7.4 by add-
ing PBS buffer (1X final). For dot blots, 15 μl of DMPC (1
mg/ml 1,2-dimyristoyl-sn-glycero-3-phosphocholine; Aventi
Polar Lipids, Inc., Alabaster, AL USA) was allowed to dry on
a PVDF transfer membrane (HybondTM-P, GE Healthcare
Europe GmbH, Orsay, France) for 15 min. The DMPC mem-
branes were then incubated at 37°C for 24 h with 2 ml of 20
μM WT or M8 proteins. Blots were washed twice with PBS
and blocked with PBS 5% dried milk (Régilait, Saint Martin
Belle Roche France). To detect the proteins bound to blotted
DMPC, Ponceau red (Sigma, St. Louis, MO USA), mouse
anti-His-tagged antibodies (Amersham, Piscataway, NJ, USA)
and SuperSignal® West Dura Pierce chemoluminescence kits
(Thermo Scientific, Perbio Science France SAS, Brebières,
France) were used.
We gratefully acknowledge Johanna Coindreau for her techni-
cal help and enthusiasm in searching for KO strains showing
changes in M8 and WT sensitivity. This work was supported by
grants from the CNRS (Programme Interdisciplinaire—Interface
Physique Chimie Biologie—Soutien à la prise de Risque) and
from the French Research National Agency ANR grant, project
ANR-06-MRAR-011-01 «AMYLOI». K.B. received fellowships
from the ANR and the Conseil Régional d’Aquitaine.
Supplementary materials can be found at:
used to separate clones of interest from false positive candidates.
The transformation mixture was plated onto both selective mini-
mal dextrose media and non-selective minimal galactose media.
The comparison between these two types of media allowed us
to eliminate three false positive families: isolates unable to grow
on minimal medium, isolates that did not undergo transforma-
tion and isolates that were unable to grow on minimal galactose
medium. Selected transformants were then replica plated onto a
selective galactose medium to induce M8 expression. Deletion
mutants presenting a growth defect were then selected and pro-
cessed via a serial dilution assay, to analyze their sensitivity to
M8 expression more precisely. Thus, all strains remained in their
exact original position during the microtiter plate transformation
protocol, such that each strain could be identified by its position
on the plate. Moreover, selected deletion mutants presenting a
growth defect could be easily identified.
Gene ontology classification. Genes identified during the
library screen and confirmed via fresh transformation of Mat
yeast strains were processed using FunSpe19 for MIPS and GO
classification. An additional GO classification for the function
and the localization was also performed (http://db.yeastgenome.
FM4-64 labeling. 1 ml of exponentially growing cells was
concentrated into 100 μl, placed on ice and incubated in the pres-
ence of 20 μM FM 4–64 (Invitrogen) for 15 min. Cells were then
washed twice with 1 ml of cold medium, resuspended in 1 ml of
medium (t0) and incubated for 45 min at room temperature.
Microscopy. Cells were washed in water and resuspended in
medium. DNA was stained by adding 2 ng/mL Hoechst 33342
to the mounting solution. Cells were observed using a DMLB
(Leica) fluorescence microscope coupled with a ColorView II
(Olympus) color camera.
Protein extraction and western blotting. Alkaline lysis
extraction was used for protein extraction. Briefly, 5 OD (l = 600
nm) units of yeast cells in exponential growth were permeabi-
lized with 500 μL of 0.185 M NaOH, 0.2% ß-mercaptoethanol.
After a 10-min incubation on ice, Trichloroacetic acid (TCA)
was added to a final concentration of 5%, and the samples were
incubated for an additional 10 min on ice. Precipitates were then
collected by centrifugation at 14,000 g for 5 min. Pellets were
dissolved in 30 μL of dissociation buffer (4% sodium dodecyl
sulfate, 0.1 M Tris-HCl pH 6.8, 4 mM EDTA, 20% glycerol, 2%
2-mercaptoethanol and 0.02% bromophenol blue) and 15 μl of
1 M Tris-base. Yeast proteins were incubated for 5 min at 100°C
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