Actin Dosage Lethality Screening in Yeast Mediated
by Selective Ploidy Ablation Reveals Links to
Urmylation/Wobble Codon Recognition and
Brian Haarer,* Lei Mi-Mi,* Jessica Cho,*,†Matthew Cortese,* Susan Viggiano,* Daniel Burke,‡
and David Amberg*,1
*Department of Biochemistry and Molecular Biology, State University of New York (SUNY) Upstate Medical University,
Syracuse, New York 13210,†Department of Biology, Syracuse University, Syracuse, New York 13244, and‡Department of
Biochemistry and Molecular Genetics, University of Virginia Medical Center, Charlottesville, Virginia 22908
ABSTRACT The actin cytoskeleton exists in a dynamic equilibrium with monomeric and filamentous states of
its subunit protein actin. The spatial and temporal regulation of actin dynamics is critical to the many functions
of actin. Actin levels are remarkably constant, suggesting that cells have evolved to function within a narrow
range of actin concentrations. Here we report the results of screens in which we have increased actin levels in
strains deleted for the ~4800 nonessential yeast genes using a technical advance called selective ploidy
ablation. We detected 83 synthetic dosage interactions with actin, 78 resulted in reduced growth, whereas in 5
cases overexpression of actin suppressed the growth defects caused by the deleted genes. The genes were
highly enriched in several classes, including transfer RNA wobble uridine modification, chromosome stability
and segregation, cell growth, and cell division. We show that actin overexpression sequesters a limited pool of
eEF1A, a bifunctional protein involved in aminoacyl-transfer RNA recruitment to the ribosome and actin
filament cross-linking. Surprisingly, the largest class of genes is involved in chromosome stability and
segregation. We show that actin mutants have chromosome segregation defects, suggesting a possible role
in chromosome structure and function. Monomeric actin is a core component of the INO80 and SWR chromatin
remodeling complexes and the NuA4 histone modification complex, and our results suggest these complexes
may be sensitive to actin stoichiometry. We propose that the resulting effects on chromatin structure can lead
to synergistic effects on chromosome stability in strains lacking genes important for chromosome maintenance.
Variousyeast genome-wide screens have been developed thattest pair-
wise genetic interactions between hypomorphic or hypermorphic al-
leles. These screens have been particularly useful in uncovering genetic
recognized are whole genome synthetic lethal screens whereby double-
mutant haploid strains are systematically constructed and analyzed for
phenotype (Costanzo et al. 2010). Alternatively, we have developed
whole-genome screening methods to identify deleterious combinato-
rial haploinsufficiencies (complex haploinsufficiency, or CHI) (Haarer
et al. 2007, 2011). Synthetic lethal and CHI screens are examples of
examining combinations of null and hypomorphic alleles. Others have
exploited overexpression (hypermorphs) in combination with null alleles
to identify synthetic dosage lethality (SDL) interactions (Measday et al.
2005) or synthetic dosage suppression interactions (Magtanong et al.
2011). The last two methods use modification of conventional synthetic
genetic array screening to introduce overexpression plasmids into ar-
rayed strains carrying deletion alleles of nonessential genes by genetic
crosses. Few screens, however, take a whole-genome approach to ex-
amine the more subtle effects of approximately twofold overexpression
Copyright © 2013 Haarer et al.
Manuscript received October 5, 2012; accepted for publication January 15, 2013
This is an open-access article distributed under the terms of the Creative
Commons Attribution Unported License (http://creativecommons.org/licenses/
by/3.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Supporting information is available online at http://www.g3journal.org/lookup/
1Corresponding author: Department of Biochemistry and Molecular Biology, SUNY
Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210.
Volume 3|March 2013|
of a particular gene, such as might occur with gene duplications, chro-
mosomal translocations, or whole-chromosome aneuploidies (e.g., Down
syndrome). Although synthetic genetic array-modified SDL screening
could be used with noninducible, low-copy-number plasmids, it requires
that strains be put through meiosis (sporulation), followed by selection
for desired, and counter-selection against nondesired, haploid progeny.
Here we used a new method called selective ploidy ablation (SPA) (Reid
et al. 2011) to screen for synthetic dosage interactions (SDIs) (Tong et al.
2004) with actin. SPA uses mating followed by chromosome destabili-
zation to introduce plasmids into the haploid deletion collection strains.
The query of our modified SDI procedure is the essential actin gene
of yeast. Actins are highly conserved in sequence and structure and are
well known to polymerize to form thin 7-nm filaments that are
organized by associated actin binding proteins into a diverse number of
highly dynamic structures that are collectively called the actin
cytoskeleton. The actin cytoskeleton is responsible for generating force
and movement in cells. The actin cytoskeleton functions in endocytosis,
exocytosis, polarized cell growth, cytokinesis, cell motility, translation
(Kim and Coulombe 2010), as well as less well-defined functions in
nuclear processes (Hofmann et al. 2004; Percipalle and Visa 2006) and
as a component of chromatin remodeling complexes (Shen et al. 2000;
Galarneau et al. 2000; Krogan et al. 2003). This functional complexity,
sensitivity to gene dosage (Wertman et al. 1992), and dynamic behavior
make actin a particularly rich target for genetic interaction analysis. We
have previously reported two genome-wide screens for genetic interac-
tions of actin. The first looked for CHI interactions between a null allele
of actin and ~4800 null alleles for the nonessential genes, identifying
~240 deleterious CHI interactions (Haarer et al. 2007). This screen
uncovered previously unappreciated functional relationships between
actin and the ribosome, the Ccr4-Not transcriptional regulation com-
plex, the ESCRT endocytic complexes, and the vacuolar ATPase, to
name a few. A second screen used a modification of the procedure
to examine the essential genes for CHI interactions with actin. This
screen identified an additional 60 CHI interactions, and in this collec-
tion of genes there was tremendous functional enrichment, in particular
for the proteasome and the TFIID complex (Haarer et al. 2011).
In this study we use SPA (Reid et al. 2011) to introduce a centro-
mere plasmid carrying the actin gene into the ~4800 nonessential gene
deletion collection. This procedure uses a donor strain that has galac-
tose inducible promoters driving transcription across all 16 centro-
meres and a counter-selectable marker (URA3) linked to the 16
centromeres. This donor strain was transformed with the ACT1 cen-
tromere plasmid, mated to deletion strains, chromosome loss was
induced on galactose, and haploidized strains were selected on
5-fluoroorotic acid medium. The strains undergo a meiosis-independent
transition from a 2N to 1N complement of chromosomes. The net
result of this induced chromosome loss is to isolate haploid deletion
strains containing the ACT1 centromere plasmid. We identified 78
deleterious SDIs and five synthetic dosage suppression interactions.
There was very high functional enrichment for genes that are novel
and were not previously known to be directly related to actin function.
Most prominently this included 14 genes involved in transfer RNA
(tRNA) modifications (the elongator and urmylation pathways) that
facilitate wobble position alternate base pairing and 19 genes involved
in chromatid cohesion and/or chromosome segregation.
METHODS AND MATERIALS
Plasmid and strain constructions
Yeast strains were grown on conventional media and strains were
constructed by standard genetic methods (Amberg et al. 2005). Genome
wide screens used robotics and methodologies described previously
(Haarer et al. 2007). Strains ysed in this study are listed in Support-
ing Information, Table S1. The yeast gene deletion collection was
obtained from EUROSCARF.
The nourseothricin resistance cassette (NATr) was removed from
plasmid p4339 (provided by C. Boone) as an ~1185-bp BamHI-
EcoRV fragment and cloned into BamHI-SmaI-cut YCp50 and
pKFW29, replacing the majority of the URA3 gene in each; the result-
ing plasmids were named pLMM1 (CEN, NAT) and pLMM3 (CEN,
NAT, ACT1), respectively. Yeast strain W8164-2B (Reid et al. 2011)
was transformed with pLMM1 and pLMM3 to generate yeast strains
LMMY1 and LMMY3, respectively.
Robotic screening by selective ploidy ablation
Robotic screening was carried out by pinning the Euroscarf non-
essential haploid deletion collection to YPD+G418 (200 mg/ml) and
the query strains LMMY1 and LMMY3 to YPD+NAT (100 mg/ml).
After 2-d growth at 25?, deletion strains were mated to each query
strain by successively pinning each to YPD plates and incubating
overnight at 25?. Mated strains were pinned to YPD+G418+NAT
and incubated at 25? for 2 d to select for diploids. Destabilization of
query strain chromosomes was induced by growth on YPGAL+G418
+NAT for 2 d at 25?, after which, strains were pinned to SC+FOA
+G418+NAT (NAT concentration was increased to 150 mg/mL in SC
media) at 30? and 37?. Relative growth of strains from LMMY1- vs.
LMMY3-based screens was noted after 2 to 4 d.
Confirmation of SDIs
Sensitivity to extra ACT1 was confirmed by independently transforming
candidate deletion strains with YCp50 and pKFW29 (YCp50-ACT1)
(Wertman et al. 1992). Strains were assessed for the subsequent
abilities of transformants to grow at various temperatures on SC-
ura medium. Growth of selected transformants were quantified by
growing in liquid SC-ura and following OD595 using a TECAN
Infinite 200 (Tecan Systems Inc., San Jose, CA) shaking incubator/
Assays for chromosome loss
Actin alleles were integrated into a disomic chromosome VII strain
5912-SD4 after EcoRI digests of plasmids carrying HIS3-marked
alleles of actin (Table S4) and transformants were selected on SC-
his medium. These strains were grown in SC-lys-tyr medium to select
for the disomes and 1022, 1023, and 1024dilutions were plated on
YPD + cycloheximide medium to select for loss of heterozygosity for
the cyh2 allele. In addition, to determine cell viability, a 1024and 1025
dilution was plated on YPD. White (ade2 ade3) and His2(ade3)
colonies were quantified on the cycloheximide plates and divided by
the numbers of viable cells in each culture to determine rates of
chromosome loss. Each strain was analyzed three independent times,
average chromosome loss frequencies and standard deviations were
calculated (Table S4).
Primary robotic screens for synthetic dosage lethal
interactions with actin
The ACT1/CEN plasmid pLMM3 and a negative control plasmid
pLMM1 were transformed into the selective ploidy ablation host
strain W8164-2B (Reid et al. 2011) and robotically mated to the Euro-
scarf haploid collection of null allele strains for the nonessential gene
set. After diploid selection, the strains were robotically transferred to
|B. Haarer et al.
galactose-containing medium to induce transcription across the cen-
tromeres of the 16 chromosomes. Galactose-induced transcription
across centromeres results in mitotic instability and can be used to
induce 2N-1 aneuploidy (Hill and Bloom 1987; Reid et al. 2008). Cells
were transferred to medium containing 5-fluoroorotic acid after
growth on galactose to counter-select the URA3 markers linked to
the 16 centromeres derived from strain W8164-2B. Selection for the
null allele on G418 media is maintained throughout to preclude loss of
the null allele through destabilization of mitotically recombined chro-
mosomes. The strains carrying the actin plasmid were scored manu-
ally/visually for colony size relative to control strains bearing pLMM1.
In contrast to the methodology of Reid et al. (2011), we screened by
using a 384-strain, rather than 1536-strain, format and used visual
overlays to compare colony sizes. In our screen, we found that colonies
were sufficiently irregular to prevent accurate digitization of colony
size; this, coupled with more extensive edge-effects from a 384-strain
format, necessitated manual analysis of data. We compared results
from three independent screens and chose for further analysis those
strains that displayed significant growth deficits relative to control in at
least two of the screens.
Confirming synthetic dosage sick or lethal interactions
The YCp-ACT1 plasmid and YCp50 were transformed in parallel into
haploid deletion strains and the transformations were scored for both
transformation efficiency and colony size upon restreaking. Figure 1
shows examples of plasmid-containing strains identified by robotic
screening that showed a range of severity of growth phenotypes in
thepresence ofthe YCp-ACT1plasmid.Theatp1Δstrainhad amodest
growth defect at 37? (Figure 1B), tpm1Δ had severe growth defects at
both temperatures (Figure 1, A and B), whereas ctk1Δ and elp2Δ had
growth defects only at 30? (Figure 1, D and E). Strains having the
strongest interactions in the transformation assay were reproducibly
identified in the robotic screens. Confirmation tests led to the identi-
fication of 25 synthetic dosage lethal or synthetic dosage sick inter-
actions. Note that we collectively refer to these negative genetic
interactions as SDL interactions.
We examined published data and data available from The Saccha-
romyces Genome Database (http://www.yeastgenome.org/) concerning
the functions of the genes showing SDI with actin and derived a list of
functionally related genes we might expect to show SDIs with actin
(the implicated genes). We tested an additional 136 genes by using the
transformation-based procedure described previously and found that
58 show an SDI (either negative or positive) with actin. The implicated
list was more enriched for actin SDIs than the entire nonessential gene
deletion set, suggesting functional enrichment in genes that show SDI
Synthetic dosage suppression interactions
Although the largest class of interactions identified was negative inter-
actions, a small number of synthetic dosage suppression interactions
(high copy suppression) were identified. The strongest suppression was
seen with a deletion of the gene for verprolin a yeast ortholog of
human WASP interacting protein or WIP. A vrp1Δ strain is unable to
grow at 37?, and this growth defect can be completely suppressed by an
extra copy of the actin gene (Figure 2, C and D). Suppression of the
vrp1-1 allele by extra actin has been previously reported (Vaduva et al.
1997). The vrp1Δ strain also grows less well at 30? than a wild-type
strain, and this defect is also suppressed by extra actin (Figure 2, A and
B). Interestingly, an actin filament stabilizing form of actin encoded by
Figure 1 SDIs with actin. atp1Δ and tpm1Δ (A, B, and C), ctk1Δ and elp2Δ (D, E, and F) strains were transformed with control plasmid YCp50 or
actin plasmid pKFW29 according to the keys (C and F), the resulting transformants were struck for single colonies, and grown at 30? (A and D) or
37? (B and E).
Volume 3 March 2013| Actin Dosage Lethality and Suppression|
the act1-159 allele (Belmont and Drubin 1998) has little-to-no ability to
suppress vrp1Δ (Figure 2; act1-159 is carried on plasmid pBH662).
Verprolin is reported to be an actin monomer binding protein that
also interacts with the Arp2/3 activator Las17 (WASP) and the cyto-
kinesis regulator Hof1p (Munn and Thanabalu 2009). Verprolin acts as
an actin monomer chaperone for actin filament assembly (Munn and
Thanabalu 2009), and we propose that loss of verprolin can be sup-
pressed by increasing the amount of monomeric actin but not filamen-
We also observed suppression of the growth defects caused by
deletion of GIM3 and PAC10, two genes for components of the pre-
foldin complex. Deletions of several genes for proteins of the prefoldin
complex were complex haploinsufficient with an actin deletion, includ-
ing gim3Δ and pac10Δ (Haarer et al. 2007). The prefoldin complex acts
as a chaperone delivering unfolded actin and tubulin to the CCT
chaperonin complex for folding (Vainberg et al. 1998). Interestingly,
act1Δ was also complex haploinsufficient with deletion of two genes for
CCT complex members, cct4Δ and cct8Δ (Haarer et al. 2011). Col-
lectively, these results are consistent with mass-action effects on actin
folding: (1) When actin levels are low simultaneous reductions in
prefoldin or CCT complex levels leads to pools of folded actin that are
inadequate to sustain cell viability, and (2) Under conditions of re-
duced folding capacity, such as in prefoldin deletion mutants, folded
actin becomes limiting, affecting growth rate, but increasing actin ex-
pression can push the equilibrium such that levels of folded actin
become adequate to restore normal growth rates.
Finally, reduced growth rates in sac6Δ and taf14/anc1Δ could be
partially suppressed by actin overexpression. Sac6 cross-links and
stabilizes actin filaments within the actin cortical patch (Goodman
et al. 2002) and we propose that in its absence, an increased actin
concentration can partially compensate for the loss of this activity.
Anc1/Taf14 is reported to be in a number of protein complexes that
are involved in transcription and chromatin remodeling, some of
these complexes also contain actin (e.g., the INO80 complex (Shen
et al. 2000)). anc1Δ/taf14Δ is CHI with act1Δ suggesting that Anc1p/
Taf14p-containing complexes are sensitive to actin stoichiometry.
The actin SDI gene network
We have found 78 genes that display SDL interactions and five genes
whose deletions are partially suppressed by an extra copy of the actin
Figure 2 Extra monomeric actin suppresses the loss of verprolin. Wild-type yeast strain BY4741 or a vrp1Δ strain were transformed with the empty
control plasmid YCp50, the YCp-ACT1 plasmid pKFW29, or the YCp-act1-159 plasmid pBH662. vrp1Δ transformants were struck on SC-Ura plates
and incubated at 30? (A) or 37? (C). BY4741 (wild-type) and vrp1Δ transformants were inoculated into SC-Ura liquid medium and growth rates were
monitored in a TECAN microplate reader at 30? (B) or 37? (D).
|B. Haarer et al.
gene (Table S2). It is unlikely that we have identified all of actin’s SDIs
for the nonessential gene set. There is some overlap between the non-
essential genes that showed a CHI interaction with actin and the genes
that show SDI with actin; these include CHS1, CNM67, SPC72, GIM3,
PAC10, BEM2, VMA4, VMA7, VMA8, VMA9, VMA10, VMA13, SAC2/
VPS52, RSM19, and SHP1. Loss of these genes causes sensitivity to both
elevated and decreased actin stoichiometry. The actin SDI network is
highly enriched for specific functional categories. Table S2 shows a list
of all genes identified in our SDI screen. Based on broad functional
relatedness, the genes are displayed in groups. The first group, shown in
green, comprises genes that in some way are related to a specific set of
tRNA uridine modifications that allow for wobble position base-pairing
during translation. The second category of genes, displayed in red, is
functionally related to chromatid cohesion, chromosome segregation,
and microtubule function. Members of the third group (shown in blue)
are involved in actin function, the cell cycle, and cell growth. We also
identified eight genes for vacuolar ATPase subunits (VMA2, VMA4,
VMA7, VMA8, VMA9, VMA10, VMA13, and VMA16), two of the three
genes that encode for subunits of the kinase that phosphorylates the C-
terminal domain of RNA Polymerase II (CTK1 and CTK2), and one
gene involved in endosome trafficking (SAC2).
The elongator complex, urmylation, and tRNA wobble
codon base pairing
The Funspec web site (http://funspec.med.utoronto.ca/) was used to
determine whether there was statistically significant functional enrich-
ment in the SDI network (Table S3). The most significant enrichment
(P = 1 · 10214) was for the Gene Ontology Biological Process “tRNA
wobble uridine modification.” We identified most of the genes for two
pathways that catalyze two modifications on the wobble anti-codon
uridine tRNA U34. The “elongator holoenzyme complex” (P value for
enrichment = 6.9 · 10210) is encoded by the genes ELP2, ELP3, ELP4,
ELP6, and IKI3 and catalyzes the cm5 (5-carboxymethyl) moiety
of the modifications ncm5U (5-carbamoyl-methyuridine), cm5U
(Jablonowski and Schaffrath 2007). The product of TRM9 gene that
was also identified in our screen catalyzes the formation of mcm5U
and cm5U (Huang et al. 2008). The protein urmylation pathway (P =
1.47 · 10214) is encoded by the genes NCS6, ELP2, UBA4, URM1,
ELP6, NCS2, and URE2 and forms a sulfur relay system that is re-
sponsible for modifying tRNA U34 at position 2 by thiolation
(Noma et al. 2009). Some of the elongator complex proteins are also
required for the thiolation modification. Urm1, which participates in
the sulfur relay reaction, is related in sequence to ubiquitin (Hoch-
strasser 2009) and can be conjugated to the lysine residues of pro-
teins; only a few substrates have been identified (Van der Veen et al.
2011), but one of them, the hydroperoxidase Ahp1, was also iden-
tified as showing an SDI with actin.
One interpretation of these data is that the SDI with these mutants
reflect an impact of actin overexpression on translation efficiency.
Many observations have suggested a role for the actin cytoskeleton in
translation (Kim and Coulombe 2010) and translation elongation
factor 1A (eEF1A) is known to be an actin binding and cross-linking
protein (Gross and Kinzy 2005). We asked whether overexpression of
eEF1A (encoded by the paralogs TEF1 and TEF2) affects the SDI of
actin with elongator and urmylation gene deletions (Figure 3). Re-
markably, overexpression of Tef1p completely suppressed the actin
SDI with elp2Δ (Figure 3A) and ncs6Δ (Figure 3B) but had no effect
on the SDI with ctf18Δ (Figure 3C). The product of the CTF18 gene is
involved in sister chromatid cohesion and has no known role in
translation. To confirm specificity of the eEF1A suppression to the
Elp and Urm pathways, we tested several additional mutants. We
found that the elp6Δ, urm1Δ, and ncs2Δ SD interactions with actin
were suppressed by eEF1A overexpression whereas the bim1Δ, kar3Δ,
swi4Δ, ctk1Δ, msy1Δ, and luv1Δ/vps54Δ SD interactions were not. The
only exception with respect to specificity for genes involved in wobble-
U modification was suppression of the chs7Δ SD interaction by eEF1A
overexpression. However, the function of CHS7 is not known and so
we currently do not know the mechanism by which eEF1A over-
expression suppresses the actin chs7Δ SD interaction. These results
suggest that the actin cytoskeleton and translation compete for a lim-
ited pool of eEF1A (see Discussion).
The SDI with ssk1Δ was due to a background
mutation in CHL1
The ssk1Δ strain from the deletion collection was quite sensitive to
actin overexpression, especially at 37?. We could not confirm the
interaction using the independently derived ssk1Δ strain BBY181.
Backcrossing and polymerase chain reaction of strains from different
genetic backgrounds indicated that the Euroscarf strain carries a bona
fide ssk1Δ allele but that there is a second mutation in the strain that is
required for the sensitivity to actin overexpression. In crosses of the
Euroscarf strain, we observed little second-division segregation of the
second mutation, indicating that the other gene was centromere
linked. To identify this second gene, we isolated several clones, after
transformation with a genomic (YCp) library, that were able to com-
plement the temperature sensitivity associated with the combination
of actin overexpression and the second mutation. Sequencing revealed
that several of these suppressing clones encompass overlapping frag-
ments that contain genes linked to the centromere of chromosome
XVI. Subcloning and retesting indicated that the second mutation in
the Euroscarf ssk1Δ strain is in CHL1, a probable DNA helicase in-
volved in sister-chromatid cohesion and genome integrity (Saccharo-
myces Genome Database; www.yeastgenome.org). CHL1 is related to
the human ChlR1 helicase of similar function and to the BRCA1-
binding helicase FANCJ (Wu et al. 2009). Polymerase chain reaction-
based recovery and sequencing of the mutant chl1 locus revealed
a single mutation in the 2586-bp coding region that results in changing
the highly conserved (among Chl1p orthologs) glycine 548 to gluta-
mate. This position corresponds to Gly569 at/near the C-terminal
boundary of the Arch domain of the FANCJ/XPD family of helicases;
this domain is proposed to wrap around single-stranded DNA (Fan
et al. 2008; Lehmann 2008; Liu et al. 2008; Wu et al. 2012). The ssk1Δ
allele present in the deletion strain does not appear to contribute to the
pKFW29 sensitivity of strains carrying the chl1G548Emutation. We
found that the Euroscarf chl1Δ strain is just as sensitive to actin over-
expression as strains carrying the chl1G548Eallele (Figure S1). Our
fortuitous identification of chl1 led us to examine genes of related
function, resulting in an expanded list of genes that display SDI with
actin (Table S2, genes in red).
Chromatid cohesion, chromosome segregation,
and microtubule-related functions
The second large cluster of functionally related genes identified in the
screen had functions related to chromosome dynamics during mitosis.
We identified SDI with genes involved in three broadly defined
processes of genome stability, sister chromatid cohesion (DCC1,
RAD61, CTF8, CTF18, TOF1, CHL1, CTF4), kinetochore function
(BIM1, IML3, CHL4, MCM21, MCM22, CTF3, CHL1, CTF19,
MCM16), mitotic motors (KAR3, CIK1) and spindle pole body func-
tion (SPC72, CNM67). Strikingly, these genes that regulate genome
stability are thought to function primarily in mitosis (Winey and
Volume 3March 2013|Actin Dosage Lethality and Suppression|
Bloom 2012), and most of the genes encode nuclear proteins. The two
spindle pole proteins are an exception as Cmn67 resides in the outer
(cytoplasmic) plaque of the spindle pole body and Spc72 is a compo-
nent of the cytoplasmic gamma tubulin complex (Helfant 2002).
The SDI with a large number of nuclear proteins required for
chromosome segregation is puzzling, given the conventional view
that actin is a cytoplasmic protein. However, actin has been shown
to be a component of the INO80 chromatin remodeling complex
(Shen et al. 2000), the NuA4 histone acetyltransferase complex
(Galarneau et al. 2000), and the SWR chromatin remodeling com-
plex (Krogan et al. 2003) and therefore actin is clearly in a position to
influence the structure and function of chromosomes. Furthermore,
all three of these complexes have been found to associate with the
kinetochores, and mutations in Arp4, a shared component of these
complexes, cause defects in kinetochore assembly (Ogiwara et al.
2007). In addition, human Arp8, a component of INO80, localizes
to mitotic chromosomes and its depletion results in chromosome
misalignment during mitosis (Aoyama et al. 2008).
To determine whether actin has a role in chromosome segregation
we analyzed a set of actin alanine scan mutants (Wertman et al. 1992)
for rates of chromosome loss in strains disomic for chromosome VII.
Figure 4A is a graphical representation of the disomic chromosome
VII used to score chromosome loss in the actin mutants. The disomic
chromosome VII is heterozygous for several alleles that can be used to
select for chromosome instability events and distinguish between
chromosome loss, mitotic recombination and gene conversion. The
actin alleles (Table S4) were integrated into the disomic strain and
cells were grown in medium selecting for the disome and plated on
medium containing cycloheximide. Resistance to cycloheximide
requires loss of the wild-type CYH2 allele, which can occur by gene
conversion, mitotic recombination, or loss of the chromosome indicated
with an asterisk in Figure 4A. Colonies that arose due to chromosome
loss were white (ade32ade22) and his2(ade32). The results of this
analysis are in Table S4. Three of the alleles were found to cause an
~10-fold increase in chromosome loss: act1-116 (D187A, K191A),
act1-117 (R183A, D184A), and act1-121 (E83A K84A). Figure 4B
shows the location of the mutated residues encoded by these alleles
on a surface rendering of the yeast actin molecule (Vorobiev et al.
2003). The most notable aspect of these data are that the mutated
residues would be buried in an actin filament and therefore inacces-
sible to actin filament binding proteins. Therefore, if these residues
identify sites of protein-protein interaction, they would most likely
indicate binding to an actin monomer, as this surface would only be
accessible within the monomer. Interestingly, there is no evidence
that the actin found in chromatin remodeling complexes is in the
filamentous form, quite to the contrary, the stoichiometries are most
consistent with the actin being in the monomer state (Shen et al.
2000; Galarneau et al. 2000; Krogan et al. 2003; Fenn et al. 2011). It
is possible that these mutations identify a binding interface with
Arp4p, which is known to interact directly with actin within chro-
matin remodeling and modification complexes (Fenn et al. 2011).
Network analysis of whole-genome genetic interaction data from yeast
indicates a small-world network with short path lengths between
genes and processes (Boone et al. 2007). This suggests a level of in-
tegration between cellular systems that we are just beginning to ap-
preciate but with little understanding of the molecular mechanisms of
integration. Our previous studies performing CHI screens with actin
identified large numbers of genes (~300) whose functions were
enriched for a number of core cellular processes (Haarer et al. 2007,
2011), which suggests that the actin cytoskeleton may be particularly
important for facilitating cross-talk and integration between cellular
functions. The actin cytoskeleton is well suited to such a role as actin
Figure 3 Tef1p overexpression suppresses the actin SD
interactions with elp2Δ and ncs6Δ but not ctf18Δ. An
elp2Δ strain (A), an ncs6Δ strain (B), and a ctf18Δ strain
(C) were transformed with the indicated combinations of
control plasmids YCplac111 and YCp50, or actin expres-
sion plasmid pKFW29 and Tef1p overexpression plas-
mid YEp-TEF1. Transformants were streaked on plates
according to the key (D) and incubated at 30?C.
| B. Haarer et al.
is multifunctional, is found in a diverse number of protein complexes
interacting with a large number of proteins, and is found in both the
cytoplasmic and nuclear compartments. Furthermore, actin can exist
in a number of different states of organization that change rapidly in
response to environmental change and stress. An excellent example of
a possible integrating role of actin is suggested by our recent obser-
vations showing that actin disruption can impact proteasome function
and vice-versa and that these two protein complexes interact directly.
This functional coupling was first suggested by CHI interactions be-
tween a null allele of actin and null alleles of many proteasome sub-
unit genes (Haarer et al. 2011).
Prominent in our CHI screens has been the identification of genes
encoding core components of the translation apparatus, in particular
ribosomal subunit genes (Haarer et al. 2007). Ribosomes are associ-
ated with actin filaments in vivo (Wolosewick and Porter 1976; Lenk
et al. 1977; Wolosewick and Porter 1977), and disruption of the actin
cytoskeleton in yeast leads to a reduction in protein synthesis (Kandl
et al. 2002; Gross and Kinzy 2007). One component of the translation
apparatus, eEF1A, has been shown to bind and cross-link actin fila-
ments and to play a role in proper actin organization in yeast (Gross
and Kinzy 2005). eEF1A’s role in translation is to recruit aminoacyl-
tRNAs to the ribosome during translation and eEF1A binding to
aminoacyl-tRNAs is inhibited by F-actin binding (Liu et al. 1996).
These results suggest that eEF1A mediates cross-regulation between
the actin cytoskeleton and protein synthesis. One of the largest groups
of genes identified in this SDI study includes genes for the urmylation
pathway and elongator complex proteins, which have an indirect role
in translation by modifying the wobble uridine in the anti-codon loop
of some tRNAs (Jablonowski and Schaffrath 2007; Noma et al. 2009).
The negative SDIs caused by actin overexpression in strains deleted
for urmylation and elongator pathway genes can be suppressed by
overexpressing eEF1A. We propose that eEF1A is limiting in cells and
this pool is partitioned between binding to the actin cytoskeleton and
being free to deliver aminoacyl-tRNAs to the ribosome. Overexpres-
sion of actin creates more binding sites for eEF1A, leaving less eEF1A
free to participate in translation elongation. When combined with
defects in tRNA uridine wobble anticodon modification, cumulative
reductions in translation elongation rates result in inadequate rates of
protein synthesis and reductions in cell growth and viability. Interest-
ingly, most proteins involved in translation are very abundant in cells
but two reports suggest that eEF1A may be present in only a few
hundred copies (Ghaemmaghami et al. 2003; Huh et al. 2003). How-
ever, an earlier report claims that eEF1A is an abundantly expressed
protein (Thiele et al. 1985) and so the expression level of eEF1A
remains unresolved at this time. Regardless, we theorize that eEF1A
levels evolved to be limiting as a mechanism for cells to coordinate
protein synthesis with the state of the actin cytoskeleton; under con-
ditions that cause F-actin disassembly bulk protein synthesis would be
increased while under conditions that cause enhanced F-actin assembly,
protein synthesis would be reduced. It was recently reported that de-
creased levels of eEF1A can increase the rapid tRNA decay pathway and
thus reduce the levels of tRNAs in the cell (Dewe et al. 2012). Therefore,
increased sequestration of eEF1A on actin filaments could also be af-
fecting rates of protein translation through reductions in tRNA levels.
Previous genetic screens with actin have uncovered a number of
connections to processes that regulate chromatin. In one of the first
noncomplementation screens performed in yeast, alleles of ANC1
(Actin Non-Complementing) were found to not complement the
act1-1 allele of actin (Welch et al. 1993). ANC1 was also identified
in our screen for complex haploinsufficiencies (a variation of a non-
complementation screen using null alleles) with an act1Δ allele
(Haarer et al. 2007). Anc1p/Taf14p, as well as actin, has since been
found to be a core component of the INO80 chromatin remodeling
complex (Shen et al. 2000, 2003). Our CHI screen with act1Δ also
identified deletion alleles of genes for three other components of
INO80 including ARP8, IES1, and IES3 (Haarer et al. 2007) and the
SDI screen described here identified IES4. In addition, the CHI screen
identified genes for three components of the NuA4 histone acetylase
complex including EAF3, EAF5, and EAF6. Actin is also known to be
a core component of the NuA4 complex (Galarneau et al. 2000). Actin
and the actin-related protein Arp4 comprise a shared core module of
not only the INO80 and NuA4 complexes but also the SWR chroma-
tin remodeling complex (Krogan et al. 2003). We propose that these
complexes and the processes they regulate are sensitive to actin gene
dosage and this is the basis for our identification of genes involved in
chromosome segregation in the SDI and CHI screens.
The INO80, SWR, and NuA4 complexes all share a common
influence on chromatin composition by regulating the localization of
histone H2A.Z-containing nucleosomes (Htz1p in yeast). NuA4 has
been shown to acetylate histone H4, and is required to recruit the
SWR chromatin remodeling complex. The function of SWR is to
Figure 4 Mutations in conventional actin lead to elevated levels of
chromosome loss. (A) Diagram of the two copies of chromosome VII in
strain 5912-SD4 used to measure the frequencies of chromosome loss
in actin mutants. Recessive, loss-of-function alleles of genes are
indicated in lower case whereas wild-type alleles of genes are
indicated in upper case. In addition, the strain has a loss of function
allele of the ade2 gene. Loss of the wild-type CYH2 allele by loss-of-
heterozygosity can be selected for by cycloheximide resistance, which
can occur by gene conversion, mitotic recombination, or chromosome
loss. Loss of the chromosome indicated by an asterisk leads to cyclo-
heximide resistant, white colonies (ade2 ade3) that require histidine
(his-due to the ade3 allele). (B) The location of mutated residues in
actin that cause chromosome loss. The solvent exposed surface of
yeast actin was rendered with the program Chimera (http://www.cgl.
et al. 2003). The side-chains of residues mutated by allele act1-116 are
rendered in red, the side chains of residues mutated by allele act1-117
are rendered in blue, and the side chains of residues mutated by act1-
121 are rendered in yellow. Note that the surface shown is largely
buried within an actin filament.
Volume 3March 2013| Actin Dosage Lethality and Suppression|
exchange histone H2A for histone H2A.Z in discreet regions of the
genome such as at double-strand breaks, promoters, heterochromatin-
euchromatin boundaries, and centromeric DNA (Lu et al. 2009). The
INO80 complex on the other hand opposes this activity by replacing
H2A.Z with H2A; in INO80 mutants H2A.Z becomes mis-localized
through-out the yeast genome (Watanabe and Peterson 2010). The
H2A vs. H2A.Z composition of histones locally affects the structure,
composition and therefore activity/function of that region of the ge-
nome. Most relevant to the screen presented here are observations
that Htz1p recruitment to centromeres is dependent on both the
NuA4 and SWR complexes and that deletion of HTZ1, and genes
for components of both the SWR and NuA4 complexes, result in
chromosome segregation defects comparable to those we report here
for actin mutants (Krogan et al. 2004).
All reports for the composition of the INO80, NuA4, and SWR
complexes indicate that actin is present as a monomer (Galarneau
et al. 2000; Shen et al. 2000; Krogan et al. 2003; Fenn et al. 2011).
Over the years, the presence of nuclear actin has been controversial in
part because it has been difficult to detect in the nucleus even with
fluorescently labeled, high affinity F-actin binding reagents such as
phalloidin. Perhaps nuclear actin functions as a monomer and its
levels are kept low to prevent polymerization from interfering with
its interactions with components of nuclear complexes such as INO80,
NuA4, and SWR. Arp4p and Arp8p have been shown to interfere with
actin polymerization in vitro (Fenn et al. 2011), suggesting that actin-
actin interactions within actin filaments are exclusive to actin-Arp
interactions. We theorize that our actin CHI screen identified genes
for components of the INO80 and NuA4 complexes because nuclear
actin levels are too low to adequately populate these complexes. Fur-
thermore, we suggest that the SDI screen with actin identified so many
genes involved in chromosome segregation because nuclear actin has
been raised to such a level that actin-actin interactions are interfering
with the actin-Arp4 interaction required for the functions of INO80,
NuA4, and SWR complexes. The result is misregulated deposition of
Htz1p at centromeres and cumulative and toxic levels of chromosome
Using SPA for performing dosage screens has limitations, as the
overlap between screens shows that the procedure produced signif-
icant numbers of false positives and negatives. The advantages of the
SPA procedure is that it allows any lab to introduce plasmids into
haploid yeast collections without requiring the liquid robotics that are
typically not available to most labs. While not exhaustive on its own,
the SPA procedure is very effective for identifying relevant pathways
for follow-up analysis.
In summary, the results presented here and in our previous CHI
screens suggest that many cellular pathways that involve actin have
evolved to operate within a narrow range of actin concentrations
coupled to a finely tuned balance between the pools of monomeric vs.
filamentous actin. A likely consequence of this situation is that many
cellular processes can be coordinately regulated by stresses that affect
the G- to F-actin ratio; some processes may be sensitive to excessive
actin monomer or excessive actin filaments, while others may be
sensitive to limitations in actin monomer or actin filaments.
We thank Charlie Boone for the gift of plasmid p4339. We thank Bob
Reid and Rodney Rothstein for sharing strain W8164-2B prior to
publication. D.A. was supported by National Institutes of Health
(NIH) grant GM074992 and D.B. was supported by NIH grant
Amberg, D. C., D. J. Burke, and J. N. Strathern, 2005
Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
Aoyama, N., A. Oka, K. Kitayama, H. Kurumizaka, and M. Harata,
2008The actin-related protein hArp8 accumulates on the mitotic
chromosomes and functions in chromosome alignment. Exp. Cell Res.
Belmont, L. D., and D. G. Drubin, 1998
reveals roles for actin dynamics in vivo. J. Cell Biol. 142: 1289–1299.
Boone, C., H. Bussey, and B. J. Andrews, 2007
tions and networks with yeast. Nat. Rev. Genet. 8: 437–449.
Costanzo, M., A. Baryshnikova, J. Bellay, Y. Kim, E. D. Spear et al.,
2010 The genetic landscape of a cell. Science 327: 425–431.
Dewe, J. M., J. M. Whipple, I. Chernyakov, L. N. Jaramillo, and E. M.
Phizicky, 2012 The yeast rapid tRNA decay pathway competes with
elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one
or more of several modifications. RNA 18: 1886–1896.
Fan, L., J. O. Fuss, Q. J. Cheng, A. S. Arvai, M. Hammel et al., 2008
helicase structures and activities: insights into the cancer and aging
phenotypes from XPD mutations. Cell 133: 789–800.
Fenn, S., D. Breitsprecher, C. B. Gerhold, G. Witte, J. Faix et al., 2011
biochemistry of nuclear actin-related proteins 4 and 8 reveals their interac-
tion with actin. EMBO J. 30: 2153–2166.
Galarneau, L., A. Nourani, A. A. Boudreault, Y. Zhang, L. Heliot et al.,
2000 Multiple links between the NuA4 histone acetyltransferase com-
plex and epigenetic control of transcription. Mol. Cell 5: 927–937.
Ghaemmaghami, S., W. K. Huh, K. Bower, R. W. Howson, A. Belle et al.,
2003Global analysis of protein expression in yeast. Nature 425: 737–741.
Goodman, A., B. L. Goode, P. Matsudaira, and G. R. Fink, 2002
cerevisiae calponin homologue Scp1 functions with fimbrin to regulate
stability and organization of the actin cytoskeleton. Mol. Biol. Cell. 14:
Gross, S. R., and T. G. Kinzy, 2005 Translation elongation factor 1A is
essential for regulation of the actin cytoskeleton and cell morphology.
Nat. Struct. Mol. Biol. 12: 772–778.
Gross, S. R., and T. G. Kinzy, 2007Improper organization of the actin
cytoskeleton affects protein synthesis at initiation. Mol. Cell. Biol. 27:
Haarer, B., S. Viggiano, M. A. Hibbs, O. G. Troyanskaya, and D. C. Amberg,
2007 Modeling complex genetic interactions in a simple eukaryotic
genome: actin displays a rich spectrum of complex haploinsufficiencies.
Genes Dev. 21: 148–159.
Haarer, B., D. Aggeli, S. Viggiano, D. J. Burke, and D. C. Amberg,
2011 Novel interactions between actin and the proteasome revealed by
complex haploinsufficiency. PLoS Genet. 7: e1002288.
Helfant, A. H., 2002Composition of the spindle pole body of Saccharo-
myces cerevisiae and the proteins involved in its duplication. Curr. Genet.
Hill, A., and K. Bloom, 1987Genetic manipulation of centromere function.
Mol. Cell. Biol. 7: 2397–2405.
Hochstrasser, M., 2009 Origin and function of ubiquitin-like proteins.
Nature 458: 422–429.
Hofmann, W. A., L. Stojiljkovic, B. Fuchsova, G. M. Vargas, E. Mavrommatis
et al., 2004Actin is part of pre-initiation complexes and is necessary for
transcription by RNA polymerase II. Nat. Cell Biol. 6: 1094–1101.
Huang, B., J. Lu, and A. S. Bystrom, 2008
genes required for formation of the wobble nucleoside 5-methoxycarbo-
nylmethyl-2-thiouridine in Saccharomyces cerevisiae. RNA 14: 2183–2194.
Huh, W. K., J. V. Falvo, L. C. Gerke, A. S. Carroll, R. W. Howson et al.,
2003Global analysis of protein localization in budding yeast. Nature
Jablonowski, D., and R. Schaffrath, 2007
and tRNase killer toxin from yeast. Biochem. Soc. Trans. 35: 1533–1537.
Kandl, K. A., R. Munshi, P. A. Ortiz, G. R. Andersen, T. G. Kinzy et al.,
2002Identification of a role for actin in translational fidelity in yeast.
Mol. Genet. Genomics 268: 10–18.
Methods in Yeast
The yeast V159N actin mutant
Exploring genetic interac-
A genome-wide screen identifies
Zymocin, a composite chitinase
|B. Haarer et al.
Kim, S., and P. A. Coulombe, 2010 Download full-text
organizer and regulator of translation. Nat. Rev. Mol. Cell Biol. 11: 75–81.
Krogan, N. J., M. C. Keogh, N. Datta, C. Sawa, O. W. Ryan et al., 2003
Snf2 family ATPase complex required for recruitment of the histone H2A
variant Htz1. Mol. Cell 12: 1565–1576.
Krogan, N. J., K. Baetz, M. C. Keogh, N. Datta, C. Sawa et al., 2004
of chromosome stability by the histone H2A variant Htz1, the Swr1 chro-
matin remodeling complex, and the histone acetyltransferase NuA4. Proc.
Natl. Acad. Sci. USA 101: 13513–13518.
Lehmann, A. R., 2008XPD structure reveals its secrets. DNA Repair
(Amst.) 7: 1912–1915.
Lenk, R., L. Ransom, Y. Kaufmann, and S. Penman, 1977
structure with associated polyribosomes obtained from HeLa cells. Cell
Liu, G., J. Tang, B. T. Edmonds, J. Murray, S. Levin et al., 1996
sequesters elongation factor 1alpha from interaction with aminoacyl-
tRNA in a pH-dependent reaction. J. Cell Biol. 135: 953–963.
Liu, H., J. Rudolf, K. A. Johnson, S. A. McMahon, M. Oke et al.,
2008 Structure of the DNA repair helicase XPD. Cell 133: 801–812.
Lu, P. Y., N. Levesque, and M. S. Kobor, 2009
chromatin-modifying complexes with overlapping functions and com-
ponents. Biochem. Cell Biol. 87: 799–815.
Magtanong, L., C. H. Ho, S. L. Barker, W. Jiao, A. Baryshnikova et al.,
2011Dosage suppression genetic interaction networks enhance func-
tional wiring diagrams of the cell. Nat. Biotechnol. 29: 505–511.
Measday, V., K. Baetz, J. Guzzo, K. Yuen, T. Kwok et al., 2005
yeast synthetic lethal and synthetic dosage lethal screens identify genes
required for chromosome segregation. Proc. Natl. Acad. Sci. USA 102:
Munn, A. L., and T. Thanabalu, 2009
sites and some very HOT prolines. IUBMB Life 61: 707–712.
Noma, A., Y. Sakaguchi, and T. Suzuki, 2009
of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA
wobble positions. Nucleic Acids Res. 37: 1335–1352.
Ogiwara, H., A. Ui, S. Kawashima, K. Kugou, F. Onoda et al., 2007
related protein Arp4 functions in kinetochore assembly. Nucleic Acids
Res. 35: 3109–3117.
Percipalle, P., and N. Visa, 2006 Molecular functions of nuclear actin in
transcription. J. Cell Biol. 172: 967–971.
Reid, R. J., I. Sunjevaric, W. P. Voth, S. Ciccone, W. Du et al.,
2008Chromosome-scale genetic mapping using a set of 16 condition-
ally stable Saccharomyces cerevisiae chromosomes. Genetics 180: 1799–
Reid, R. J., S. Gonzalez-Barrera, I. Sunjevaric, D. Alvaro, S. Ciccone et al.,
2011 Selective ploidy ablation, a high-throughput plasmid transfer
protocol, identifies new genes affecting topoisomerase I-induced DNA
damage. Genome Res. 21: 477–486.
Emerging role for the cytoskeleton as an
NuA4 and SWR1-C: two
Verprolin: a cool set of actin-binding
Shen, X., G. Mizuguchi, A. Hamiche, and C. Wu, 2000
modelling complex involved in transcription and DNA processing. Na-
ture 406: 541–544.
Shen, X., R. Ranallo, E. Choi, and C. Wu, 2003
proteins in ATP-dependent chromatin remodeling. Mol. Cell 12: 147–155.
Thiele, D., P. Cottrelle, F. Iborra, J. M. Buhler, A. Sentenac et al.,
1985Elongation factor 1 alpha from Saccharomyces cerevisiae. Rapid
large-scale purification and molecular characterization. J. Biol. Chem.
Tong, A. H., G. Lesage, G. D. Bader, H. Ding, H. Xu et al., 2004
mapping of the yeast genetic interaction network. Science 303: 808–813.
Vaduva, G., N. C. Martin, and A. K. Hopper, 1997
is a polarity development protein required for the morphogenesis and
function of the yeast actin cytoskeleton. J. Cell Biol. 139: 1821–1833.
Vainberg, I. E., S. A. Lewis, H. Rommelaere, C. Ampe, J. Vandekerckhove
et al., 1998Prefoldin, a chaperone that delivers unfolded proteins to
cytosolic chaperonin. Cell 93: 863–873.
Van der Veen, A. G., K. Schorpp, C. Schlieker, L. Buti, J. R. Damon et al.,
2011 Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-
directed protein modifier. Proc. Natl. Acad. Sci. USA 108: 1763–1770.
Vorobiev, S., B. Strokopytov, D. G. Drubin, C. Frieden, S. Ono et al.,
2003The structure of nonvertebrate actin: implications for the ATP
hydrolytic mechanism. Proc. Natl. Acad. Sci. USA 100: 5760–5765.
Watanabe, S., and C. L. Peterson, 2010
remodeling enzymes: regulators of histone variant dynamics. Cold Spring
Harb. Symp. Quant. Biol. 75: 35–42.
Welch, M. D., D. B. Vinh, H. H. Okamura, and D. G. Drubin, 1993
for extragenic mutations that fail to complement act1 alleles identify
genes that are important for actin function in Saccharomyces cerevisiae.
Genetics 135: 265–274.
Wertman, K. F., D. G. Drubin, and D. Botstein, 1992
analysis of the yeast ACT1 gene. Genetics 132: 337–350.
Winey, M., and K. Bloom, 2012 Mitotic spindle form and function. Ge-
netics 190: 1197–1224.
Wolosewick, J. J., and K. R. Porter, 1976
croscopy of whole cells of the human diploid line, WI-38. Am. J. Anat.
Wolosewick, J. J., and K. R. Porter, 1977
heterogeneity of WI-38 cells. Am. J. Anat. 149: 197–225.
Wu, Y., A. N. Suhasini, and R. M. Brosh Jr., 2009
FANCJ-like helicases to the block of genome stability maintenance pro-
teins. Cell. Mol. Life Sci. 66: 1209–1222.
Wu, Y., J. A. Sommers, I. Khan, J. P. de Winter, and R. M. Brosh Jr.,
2012Biochemical characterization of Warsaw breakage syndrome
helicase. J. Biol. Chem. 287: 1007–1021.
A chromatin re-
Involvement of actin-related
The INO80 family of chromatin-
Stereo high-voltage electron mi-
Observation on the morphological
Welcome the family of
Communicating editor: B. J. Andrews
Volume 3March 2013| Actin Dosage Lethality and Suppression|