A Synthetic Lethal Screen Identifies a Role for the Cortical Actin Patch/Endocytosis Complex in the Response to Nutrient Deprivation in Saccharomyces cerevisiae

Corresponding author: Peter E. Sudbery, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom., p.sudbery{at}sheffield.ac.uk (E-mail)

Communicating editor: B. ANDREWS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Saccharomyces cerevisiae whi2 cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2, siw14, fen2, and thr4 mutants. Furthermore, whi2 and siw14 mutants produce large actin clumps in stationary phase similar to those seen in prk1 ark1 mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1 strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161, sla1, sla2, vrp1, ypt51, ypt52, and end3 mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2 mutants.

TO achieve balanced growth and proliferation it is necessary that cells cease division when there are insufficient nutrients. When cells of the budding yeast Saccharomyces cerevisiae are starved of nutrients, they arrest in the G₁ phase of the cell cycle in an unbudded, phase-bright state (PRINGLE and HARTWELL 1981 ). A series of physiological changes occurs, which allows cells to survive adverse environmental conditions (SNOW 1966 ; SCHENBERG-FRASCINO and MOUSTACCHI 1972 ; DEUTCH and PARRY 1973 ; PARRY et al. 1976 ; LILLIE and PRINGLE 1980 ). Cells carrying a whi2 mutation fail to show this response (SUDBERY et al. 1980 ; SAUL et al. 1985 ). As whi2 cells approach stationary phase, cell division continues beyond the point where it ceases in wild-type cells and is accompanied by prolonged expression of the G₁ cyclins Cln1 and Cln2 (RADCLIFFE et al. 1997 ). As a result of continued division without growth, whi2 cells are abnormally small in stationary phase. Furthermore, they fail to acquire the stress resistance shown by wild-type cells (SAUL et al. 1985 ). Exponentially growing whi2 cells also show increased sensitivity to certain environmental stresses, such as caffeine and 1.0 M NaCl (BINLEY et al. 1999 ).

Recently it has been shown that Whi2 acts in the stress-response pathway (KAIDA et al. 2002 ). When whi2 cells are exposed to 37°, 0.4 M NaCl, or 0.4 mM H₂O₂, the expression of stress-response genes mediated by stress-response elements (STREs) in their promoters was reduced to 50% of wild-type levels (KAIDA et al. 2002 ). STRE-mediated gene expression is also induced as wild-type cells enter stationary phase, but induction is delayed by several hours in whi2 cells and cell division continues (KAIDA et al. 2002 ). Whi2 physically interacts with Psr1, one of a pair of redundant phosphatases located in the plasma membrane (KAIDA et al. 2002 ). A psr1 psr2 mutant has phenotypes similar to those of whi2 with respect to sensitivity to Na⁺ ions and small size in stationary phase. Furthermore, expression of STRE-mediated stress-response genes is also reduced in a psr1 psr2 mutant. In addition to Psr1, Whi2 also physically interacts with Msn2, a transcription factor that plays a key role in the regulation of stress-response genes (MARTINEZ-PASTOR et al. 1996 ). Msn2 is hyperphosphorylated in whi2 or psr1 psr2 mutants (KAIDA et al. 2002 ) and overexpression of MSN2 rescues the heat sensitivity of whi2 or psr1 psr2 mutants (KAIDA et al. 2002 ). While Psr1 and Psr2 are cell surface proteins (SINIOSSOGLOU et al. 2000 ), Msn2 shuttles between the nucleus and cytoplasm (GORNER et al. 1998 ). Whi2 interacts with both Msn2 and Psr1/2 and is located throughout the cell (KAIDA et al. 2002 ). These observations suggest that Whi2 and Psr1/2 are functional partners that regulate the activity of Msn2 through its state of phosphorylation (KAIDA et al. 2002 ).

In this article, we describe the use of a colony-sectoring assay to isolate mutants that have a more severe effect on fitness when combined with a whi2 mutation. Seven independent mutants that affect a variety of cell functions were recovered. None of the mutations were strictly colethal with whi2, so we have called the mutants siw (synthetic interaction with whi2). We show that whi2 and several of the siw mutants have defects in actin organization and endocytosis. Further, we demonstrate a genetic interaction between SIW14 and ARK1 and PRK1, which encodes a pair of redundant protein kinases that regulate the stability of actin cortical patches. Finally, we show that mutations known to cause defects in the actin cytoskeleton and in endocytosis fail to show cell cycle arrest upon nutrient deprivation. Thus, we conclude that the normal function of the actin cytoskeleton and endocytosis are required to coordinate cell proliferation with nutrient availability.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and culture conditions:
Strains used in this article are described in Table 1. They were routinely cultured at 30° on YEPD [1% Difco yeast extract (Becton Dickinson, Sparks, MD), 2% Difco peptone, plus 2% glucose]. Plates were solidified by the addition of 2% Difco agar. Cells were grown to stationary phase as follows: 2.5 ml of YEPD in a 50-ml conical flask was inoculated with yeast from a fresh YEPD plate culture and incubated for 48 hr at 26° on a rotary shaker at a speed of 150 rpm. By this time, wild-type cells have arrested as unbudded cells that have a bright appearance when examined by phase-contrast microscopy. The high ratio of flask size to culture volume is important for the development of the whi2 phenotype, which is manifested only under conditions of vigorous aeration (RAHMAN et al. 1988 ). Cell volume was measured with a ZB1 Coulter Counter and Channelyser as described previously (SUDBERY et al. 1980 ). Values reported are the median of the volume distribution.

Cloning and characterization of SIW genes:
Because of the similarity of the siw mutants to mutants affecting the PKC1/SLT2 mitogen-activated protein (MAP) kinase pathway, siw mutants were transformed with plasmids carrying wild-type copies of all the genes in this pathway. The mutant originally designated siw9 was complemented by a centromeric plasmid carrying the SLT2 gene, suggesting that siw9 is an allele of SLT2. This was confirmed by the observation that siw9 failed to complement an slt2 mutation. This mutation is referred to as slt2^siw9. The remaining mutants were transformed with a genomic library constructed in YCp50, a centromeric URA3 vector (ROSE et al. 1987 ; purchased from the ATCC, http://www.atcc.org/). Ura⁺ transformants, representing approximately five-genome equivalents, were replica plated onto YEPD plates containing 5 mM caffeine, and transformants that grew were retained for further study. Normally the clones that grew produced red sectors, indicating that there was no longer selection against the loss of plasmid pPR14 (ADE2 WHI2) used in the colony-sectoring screen. The dependency of caffeine-resistant growth on the presence of a plasmid from the library was examined by growing transformants on media containing 5'-fluroorotic acid (5'-FOA) that selects for cells that have lost the YCp50-based plasmid containing the URA3 gene (BOEKE et al. 1984 ). Colonies that grew on 5'-FOA were no longer able to grow on a YEPD plate containing 5 mM caffeine. Plasmids were recovered from independent colonies by transformation of Escherichia coli with total yeast DNA preparations. Recovered plasmids were transformed into the original siw mutant. Plasmids that both induced sectoring of pPR14 and conferred wild-type levels of caffeine resistance were retained for further study. Two primers (Table 2) were used to sequence 400 bp at either end of the genomic insert in the plasmids recovered. These sequences were compared to the S. cerevisiae genome database (http://genome-www.stanford.edu/Saccharomyces/) and the intervening sequence was retrieved. Subcloning was carried out using the derived restriction map of the insert to identify the minimum complementing region.

Gene deletions were carried out in two different strains: the Y763 strain used as the parent for the colony-sectoring assay (COSTIGAN et al. 1992 ) and W303a, which is known to be ssd1-1d (CVRCKOVA et al. 1995 ). Deletions were carried out as follows. FEN2 (SIW 1) was deleted using a URA3-based disruption cassette kindly supplied by G. Lucchini. The deletion was verified by Southern hybridization. For ALG9 (SIW5), ZDS1 (SIW7), THR4 (SIW12), and SIW14 (YNLO32w), pairs of PCR primers were designed (Table 2) to amplify a URA3 template so that the resulting DNA molecule consisted of the URA3 gene flanked by 45 bp of DNA homologous to the sequence immediately upstream of the start codon and downstream of the stop codon of the gene to be deleted. In the case of SIW14 (YNLO32w), BamHI and HindIII restriction sites were incorporated into the primers between the YNLO32w and URA3 sequences to aid subsequent Southern analysis of putative disruptants. PCR amplification of the URA3 template was performed for 35 cycles with an annealing temperature of 57° and the resulting PCR fragment was used to transform yeast strains Y763 and W303a, both of which are ura3. Deletion of the target open reading frame was verified by Southern hybridization or PCR analysis.

In all cases, the deleted strain was more sensitive to caffeine than was the parent. A diploid was formed by crossing the disruptant and the original mutant. Allelism was demonstrated by noncomplementation of mutant phenotypes in the diploid and 4:0 segregation of caffeine sensitivity in tetrads dissected after sporulation. The deleted strain was also crossed to a strain with the wild-type SIW allele and tetrads were dissected. In all cases, caffeine sensitivity cosegregated with the auxotrophic marker used to engineer the deletion.

Characterization of the slt2^siw9 mutation:
PCR primers (Table 2) were designed to amplify the locus from the slt2^siw9 mutant in two segments with an overlapping region spanning the SacI site at nucleotide 600, each fragment containing a BglII or HindIII site present in the respective 5' and 3' flanking regions. The resulting PCR products were digested with either BglII and SacI (5' fragment) or HindIII and SacI (3' fragment) and ligated to pUC19 digested with HindIII and BglII. The full-length sequence of the resulting slt2^siw9 clone was determined and compared to the wild-type sequence.

Mutagenesis of SIW14:
Mutagenesis of SIW14 cloned with its own promoter into the multicopy plasmid pYES2 (Invitrogen, San Diego) was carried out using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Oligonucleotides used are listed in Table 2. Following mutagenesis, the entire SIW14 gene was resequenced for each mutant allele generated.

Western blotting:
Cells were disrupted with glass beads and total soluble protein was prepared as described (BOYNE et al. 2000 ). Proteins were fractionated by SDS/polyacrylamide gel electrophoresis and electroblotted to a Hybond C nitrocellulose membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). Primary antibody was rabbit polyclonal anti-phospho-p44/42 MAP kinase (Thr202/Tyr204; Cell Signaling Technology, Beverly, MA) used at 1:1000 dilution. Two rounds of antibody binding were used to amplify the weak signal that resulted from this primary antibody. Secondary antibody was mouse anti-rabbit immunoglobulins (Jackson Immunoresearch, Cambridge, UK) used at 1:3500 dilution. Tertiary antibody was goat anti-mouse immunoglobulins conjugated to horseradish peroxidase (Dako A/S, Glostrup, Denmark) diluted 1:5000. Binding of tertiary antibody was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech) and recorded by a Gene Gnome chemiluminescence recording system (Syngene, Cambridge, UK).

Glycogen staining:
Cells from 1 ml of stationary-phase cultures were collected by centrifugation and resuspended in 1 ml of 0.2% w/v iodine and 0.4% w/v potassium iodide. After 5 min, cells were collected by centrifugation and washed 3x in distilled water and finally resuspended in 0.2 ml of water. Each sample was placed in a well of a 96-well microtiter plate and photographed with transmitted light. Cells producing glycogen stained dark purple, while cells not producing glycogen remained light brown.

Actin staining:
Cultures were stained with phalloidin conjugated to tetramethylrhodaminyl-isothiocyanate (TRITC) conjugated (Sigma, St. Louis) as previously described (ADAMS and PRINGLE 1983 ).

Heat shock:
Stationary-phase cultures of 500 µl were heated using a Hybaid thermocyler for 10 min. Samples were diluted in triplicate and plated on YEPD and the mean number of colonies that grew after 3 days was recorded. For control cultures, unheated samples of the same cultures were diluted and plated in triplicate. Viability is expressed as percentage viability of the heated cultures relative to control.

Plate dilution tests:
Cells were harvested from a freshly grown YEPD plate and resuspended in YEPD to an OD₆₀₀ of 8.0. This suspension was then serially diluted and 5 µl of the undiluted and 10^-1, 10^-2, 10^-3, and 10^-4 dilutions were spotted onto the surface of the agar plate and cultures were incubated for 3 days at 30°. These dilutions resulted in 20 cells in the spot from the most diluted suspension. All growth tests were carried out using YEPD media supplemented as indicated except for the 37° growth test in which minimal medium was used.

Lucifer yellow uptake assays:
Lucifer yellow assays were carried out as described (DULIC et al. 1991 ).

Immunocytofluorescence:
Immunocytofluorescence was carried out as described (AYSCOUGH and DRUBIN 1998 ). Mouse anti-actin antibody was purchased from Sigma and used at a dilution of 1:200. Rabbit polyclonal anti-Cdc11 was purchased from Santa Cruz and used at 1:250 dilution. Mouse polyclonal antisera to Cof1, Sac6, and Abp1 were a kind gift from Kathryn Ayscough.

Microscopy:
For differential interference contrast (DIC) and fluorescence microscopy, cells were examined with a Leica DMLB fluorescence microscope. Digital images were recorded by a low-temperature CCD camera (model RTE Princeton Instruments, Princeton, NJ) controlled by an Apple Macintosh G4 computer running Open Lab software, version 2.2.5 (Improvison, Warwick, UK). Images were exported as tif files and edited for contrast and brightness in Adobe Photoshop, version 5.5. Composite figures were assembled using Microsoft Power Point 2000.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A colony-sectoring assay identifies seven genes that interact with whi2:
To identify genes that may interact with WHI2, we carried out a colony-sectoring assay for mutations that either are colethal or show an enhanced growth defect with the whi2 allele. For this screen, we applied the methodology used by Costigan and Snyder (COSTIGAN et al. 1992 ). Briefly, the screen is based on a whi2 ade2-1 host that harbors an unstable plasmid carrying a wild-type copy of WHI2. Mutations that have a more severe phenotype in conjunction with whi2 result in selection against loss of the WHI2 plasmid and therefore produce homogeneous white colonies that lack red sectors. In this way we isolated seven mutants, which subsequent analysis showed affected the following genes: ALG9, FEN2, PRS3, SIW14, SLT2 (MPK1), THR4, and ZDS1 (see MATERIALS AND METHODS).

SIW14 encodes a protein that contains the sequence IHCNRGKHRTGCL, which is an exact match to the canonical sequence for tyrosine phosphatases, [LIVMF]HCxxGxxx[STC][STAG]x[LIVMFY], listed in the Prosite database (http://ca.expasy.org/prosite/). SLT2 (MPK1) encodes the MAP kinase of the PKC1 MAP kinase pathway (TORRES et al. 1991 ; LEE et al. 1993 ). We refer to the allele isolated in our screen as slt2^siw9. We cloned slt2^siw9 by PCR and showed that the mutation was E225K, an amino acid substitution in the conserved region IX of the kinase domain. FEN2 was first identified in a screen for mutants resistant to fenproprimorph, an inhibitor of ergosterol biosynthesis (MARCIREAU et al. 1996 ) and it was subsequently shown that Fen2 is the membrane pantothenate transporter (STOLZ and SAUER 1999 ). PRS3 is one of a family of five genes that encodes phosphoribosyl pyrophosphate synthetase, an enzyme that catalyzes the first step in a variety of biosynthetic pathways, including amino acid and nucleotide biosynthesis (CARTER et al. 1994 , CARTER et al. 1995 ). We have previously published a detailed characterization of prs3 (BINLEY et al. 1999 ). ALG9 encodes a mannosyl transferase that transfers core oligosaccharides from the lipid carrier dolichol pyrophosphate to the asparagine residues of secreted proteins in the endoplasmic reticulum (BURDA et al. 1996 ). THR4 encodes threonine synthetase, required for threonine biosynthesis. ZDS1 has been isolated in many genetic screens, hence its name, which is derived from "zillion different screens" (BI and PRINGLE 1996 ; YU et al. 1996 ). Deletion of both ZDS1 and ZDS2 genes results in a mitotic delay and elongated buds with constrictions where cytokinesis has been attempted but not completed (YU et al. 1996 ; MIZUNUMA et al. 1998 ).

Deletion phenotype of genes identified in the colony-sectoring assay:
ALG9, FEN2, PRS3, SIW14, THR4, and ZDS1 were deleted in two strain backgrounds: Y763, the host strain for the synthetic lethal screen, and W303a, a strain carrying the ssd1-1d allele in which we found that the stress sensitivity of the whi2 phenotype is enhanced (see Table 3). Strains with a slt2 allele in two different backgrounds were obtained elsewhere (Table 1). We carried out appropriate crosses to congenic strains containing a whi2 allele to determine whether the deletion alleles were colethal with whi2. In each case, viable double mutants could be constructed, showing that the mutations were not colethal with whi2. However, upon reintroduction of the unstable WHI2-bearing plasmid into each of the double mutants, it was either absolutely stable or lost at a very low frequency compared to a wild-type host. Often, where a red sector appeared it clearly grew more slowly than the rest of the colony (data not shown). Moreover, in some cases enhanced phenotypes were observed in the double mutant. For example, a fen2 whi2 mutant was unable to grow on minimal medium and an slt2 whi2 strain had a grossly abnormal morphology (data not shown). Thus, although not colethal, these mutations have a more severe phenotype when combined with whi2. For this reason we designated these mutants siw (synthetic interaction with whi2).

View this table: In this window In a new window   Table 3. Sensitivity of Y763, whi2, and siw mutants to various conditions

Most of the siw mutants showed stress sensitivities that were similar to those of a whi2 strain. Most were hypersensitive to caffeine and 1 M NaCl (Table 3). In addition, alg9, slt2^siw9, and zds1 mutants were temperature sensitive on minimal medium; fen2 and slt2^siw9 mutants were hypersensitive to 0.7 M CaCl₂; the fen2 mutant was sensitive to Calcofluor white, a characteristic of cell wall mutants; siw14 and slt2^siw9 were unable to utilize glycerol; and, finally, siw14 cells were resistant to 6.6 mM MnCl₂, which inhibits the growth of wild-type cells.

When grown to stationary phase in YEPD, siw14, fen2, and slt2 arrested as phase-dark, budded cells, which failed to accumulate glycogen (Fig 1). Moreover, siw14 cells were hypersensitive to a 53° heat shock (Table 3). Thus, like whi2, these mutants failed to have the normal physiological responses to nutrient limitation. There was no reduction in cell size. However, many of the mutants showed very large vacuoles and it is possible that the cytoplasmic mass is less than that of a wild-type cell. This is particularly marked in the case of the slt2 mutant where, interestingly, some very small cells are evident (arrows in Fig 1). In contrast, alg9, thr4, and zds1 mutants showed the normal responses to nutrient limitation (data not shown). However, the alg9 and zds1 alleles isolated in the colony-sectoring screen did arrest as phase-dark budded cells in stationary phase (data not shown). Thus, it is possible that the mutant alleles isolated in the screen have a different phenotype from that of the deletion alleles. However, the genetic complexity of the original mutants makes such a conclusion uncertain and we did not pursue this matter further.

View larger version (109K): In this window In a new window Download PPT slide   Figure 1. Stationary-phase appearance of wild-type, whi2, and siw mutants. Cells with the indicated genotype were grown in YEPD for 2 days at 30°. The median cell volumes were determined and the appearance of cells was recorded using phase-contrast optics with a x100 objective. Arrows show small cells in the slt2 strain. In the glycogen test, a positive result is indicated by a dark purple color while lack of accumulation results in light brown; these colors reproduce in the image shown here as dark gray/black and light gray, respectively. The strains grown were congenic to the wild-type W303a strain shown here. Y763, the parent strain for the synthetic lethal screen, and the respective siw deletions in the Y763 background, had a similar appearance. Bar, 10 µm.

Cytoskeletal abnormalities in stationary-phase whi2 and siw14 cells:
We previously reported that whi2 cells contain abnormal actin clumps in stationary phase (BINLEY et al. 1999 ). We examined the siw mutants for any abnormalities in the actin cytoskeleton. While the actin distribution of growing cells was normal, we observed that stationary-phase siw14 cells had a single intensely staining clump of actin in >95% of the cells examined (Fig 2A). The clumps also contained other actin-associated proteins such as Abp1, Sac6, and Cof1 (Fig 2B). This abnormality was present in all strain backgrounds containing the whi2 and siw14 mutations. We also observed that siw14 stationary-phase cells contained abnormal clumps of the septin Cdc11, which is normally located in a ring structure at the bud neck (Fig 2C).

View larger version (38K): In this window In a new window Download PPT slide   Figure 2. Siw14 mutants in stationary phase show defects in the actin and septin cytoskeletons. Wild-type (W303a) and siw14 cells were grown in exponential or stationary phases as indicated. (A) Actin visualized by immunocytofluorescence using a mouse anti-actin antibody. (B) The indicated proteins visualized by immunocytofluorescence using mouse antisera (a kind gift from Kathryn Ayscough). (C) The septin cytoskeleton visualized by immunocytofluorescence using a polyclonal antibody to Cdc11. Arrows show normal septin ring structures. Arrowheads show abnormal septin bars in stationary-phase cells. Bar, 10 µm.

Endocytosis is defective in whi2, fen2, thr4, and siw14 mutants:
Defects in the cortical actin cytoskeleton are often associated with defects in endocytosis. To determine if fluid-phase endocytosis was functioning normally in whi2 and siw mutants, we carried out lucifer yellow uptake assays. The whi2, fen2, thr4, and siw14 mutants were defective in fluid-phase endocytosis (Fig 3). The defect in fen2 mutants was particularly profound.

View larger version (79K): In this window In a new window Download PPT slide   Figure 3. Whi2, siw14, fen2, and thr4 are defective in fluid-phase endocytosis. Wild-type (W303a) cells and the indicated mutants were tested for fluid-phase endocytosis using the lucifer yellow uptake assay. Bar, 10 µm.

SIW14 interacts with ARK1 and PRK1:
Ark1 and Prk1 are two homologous protein kinases that are thought to regulate the association of the actin cortical patch complex with the endocytic machinery by phosphorylating Pan1 and Sla1 (COPE et al. 1999 ; ZENG and CAI 1999 ; ZENG et al. 2001 ). The actin clumps in whi2 and siw14 mutants are strikingly similar to the actin clumps seen in ark1 prk2 cells (COPE et al. 1999 ). Because of this phenotypic similarity, we searched for interactions between ARK1 and PRK1 with WHI2 and SIW14. We found that overexpression of SIW14 from a GAL1 promoter on a multicopy plasmid was lethal in a prk1 strain and reduced growth in an ark1 strain (Fig 4A). A high proportion of cells with no visible actin patches or cables accumulated within 4 hr upon induction of SIW14 in a prk1 strain (Fig 4B).

View larger version (111K): In this window In a new window Download PPT slide   Figure 4. Overexpression of SIW14 is lethal in a prk1 mutant. (A) Wild-type (BY742) prk1 and ark1 mutants were transformed with either the multicopy pYES2 vector or pYES2 containing SIW14 under the control of the GAL1 promoter. Dilutions were plated on YEPD or YEPGal to induce the Gal1 promoter. (B) Prk1 cells transformed with either multicopy SIW14 under the control of the GAL1 promoter in pYES2 or the pYES2 vector alone. Transformed cells were grown to mid-exponential phase on YEP-raffinose to which 1% galactose was then added and the cells were incubated for another 4 hr. Actin was visualized using phalloidin-TRITC staining. In each case, the DIC image shows the same field of cells as those stained with phalloidin. Note that the majority of pYES2-SIW14-transformed cells in the DIC field do not stain with phalloidin.

Mutants in endocytosis and the actin cytoskeleton are defective in cell cycle arrest in stationary phase:
The inability of whi2 and the siw mutants to arrest the cell cycle upon nutrient limitation could be a consequence of the defects in the actin cytoskeleton and endocytosis. To test this hypothesis, we determined whether mutations in genes known to function in endocytosis and the actin cytoskeleton have defects in cell cycle arrest. We monitored the effect on cell size and appearance in the stationary phase of deletion alleles of the following genes that have defects in actin organization or are listed in the Saccharomyces Genome Database as having defects in endocytosis: AKR1, ARK1, ARL1, CLC1, DNM1, END3, ENT1, ENT2, PKH1, PRK1, SWA2, RVS161, RVS167, SLA1, SLA2, TLG2, VAN1, VPS 4, VPS34, VRP1, YPK1, YPK2, VPS21 (YPT51), YPT52, and YPT53. Of these, we found that end3, rvs161, sla1, vrp1, sla2, vps21 (ypt51), and ypt52 arrested in stationary phase as phase-dark budded cells, instead of the phase-bright unbudded appearance of wild-type cells (Fig 5). Furthermore, vrp1 and rvs161 cells were much smaller than wild-type cells: 52 and 65%, respectively, of the size of wild-type cells. The ark1 and prk1 mutants showed some indication that cell cycle arrest was not normal, but this was not so marked and an ark1 prk1 double mutant did not show a more severe defect in nutrient arrest (data not shown). All the other mutants listed had a similar appearance and size to wild-type cells except for the akr mutant, which as noted before has an unusual "peanut" shape (PRYCIAK and HARTWELL 1996 ).

View larger version (107K): In this window In a new window Download PPT slide   Figure 5. Actin cortical patch/endocytosis mutants fail to show the normal response to nutrient deprivation. Cells with the indicated genotype were grown to stationary phase, photographed, and median cell volumes were determined as described in Fig 1. The wild-type strain shown here is BY742 since it is the parent for all strains except vps51 and ypt52, which are congenic to W303a. W303a has an identical appearance to BY742 (see Fig 1).

We examined end3, rvs161, sla1, sla2, vrp1, vps21 (ypt51), and ypt52 mutants to see if they also showed clumps of actin similar to that seen in whi2 and siw14 cells. Fig 6 shows that end3, sla1, prk1, and ypt52 did indeed show such clumps. Thus, this type of actin disorganization is a feature of cells that are unable to respond appropriately to nutrient deprivation. Clumps of actin in sla1 mutants have been reported previously (HOLTZMAN et al. 1993 ).

View larger version (76K): In this window In a new window Download PPT slide   Figure 6. Actin cortical patch/endocytosis mutants accumulate actin clumps in stationary phase. Wild-type (WT) and the indicated mutants were grown to stationary phase as described in Fig 1 and actin was visualized by rhodamine-phalloidin staining.

SIW14 shows a genetic interaction with SLT2:
The phenotypes of whi2 and siw mutants and mutants affecting the PKC1/Slt2 MAP kinase share the following phenotypic abnormalities: (a) they are sensitive to caffeine and other stresses, and these sensitivities are rescued by 1 M sorbitol and exacerbated by a ssd1-1d allele; (b) they fail to show a normal response to nutrient limitation; and (c) they display defects in the actin cytoskeleton, which in slt2 mutants is marked by loss of polarity and the appearance of actin bars in the cytoplasm (COSTIGAN et al. 1992 , COSTIGAN et al. 1994 ; MAZZONI et al. 1993 ). Moreover, we isolated an allele of SLT2 in the synthetic lethal screen. We therefore searched for interactions between the siw mutants and Slt2. We found that SIW14 on a multicopy plasmid could rescue the caffeine (Fig 7), but not the temperature sensitivity of the slt2^siw9 mutation (data not shown). The minimum inhibitory concentration of caffeine of slt2^siw9 is 4 mM, whereas that of a congenic slt2 strain is 2 mM. It is therefore likely that the slt2^siw9 kinase retains some activity. Interestingly, multicopy SIW14 exacerbated the caffeine sensitivity of the congenic slt2 strain, so that growth on 1 mM caffeine was prevented (Fig 7). Thus, multicopy SIW14 interacts with SLT2 in an allele-specific fashion: it rescues the caffeine sensitivity of the slt2^siw9 allele but enhances the caffeine sensitivity of an slt2 allele. To determine whether the putative tyrosine phosphatase active site was required for the interaction, we generated the following point mutations in three conserved residues in the putative tyrosine phosphatase active site: C214S, G217A, and R220K. The C214S mutation changes a critical cysteine residue that participates in catalysis and is known to abolish tyrosine phosphatase activity (JOHNSON et al. 1992 ). Multicopy plasmids carrying these catalytically dead versions of Siw14 were unable to rescue the caffeine sensitivity of slt2^siw9 and failed to enhance the caffeine sensitivity of slt2 (Fig 7). Thus, the interaction between Siw14 and Slt2 depends on a functioning tyrosine phosphatase active site. Surprisingly, the mutants were still able to rescue the caffeine sensitivity of an siw14 mutation (Fig 7), indicating that the tyrosine phosphatase active site is not required for at least some of the normal functions of Siw14.

View larger version (58K): In this window In a new window Download PPT slide   Figure 7. Effect of overexpressing wild-type and mutant SIW14 on the caffeine sensitivity of different slt2 mutants. The top two rows show dilutions of wild-type (W303a) or mutant strains. The concentration of caffeine and the identity of the mutant are indicated at the bottom. The remaining rows show dilutions of the particular mutant strain transformed with wild type or the indicated mutants of SIW14, cloned with its own promoter into the 2-µm-based pYES2 plasmid.

Slt2 is activated by dual phosphorylation on threonine 190 and tyrosine 192 (LEE et al. 1993 ). Since Siw14 is a putative tyrosine phosphatase, it is possible that multicopy SIW14 affects the phosphorylation state of Slt2. To determine if this were the case, we used an antibody specific to the activated form of Slt2 to monitor the phosphorylation state of Slt2 (MARTIN et al. 2000 ). Phosphorylation of Slt2 increased upon exposure to caffeine as reported previously (Fig 8; MARTIN et al. 2000 ). Although the level of activating phosphorylation was reproducibly reduced in an slt2^siw9 mutant upon caffeine exposure, perhaps explaining its reduced activity, the level of phosphorylation was unaffected by multicopy SIW14. Furthermore, the level of phosphorylation of wild-type Slt2 was also unaffected by deletion or overexpression of SIW14. Thus, Siw14 does not act directly to dephosphorylate Slt2.

View larger version (21K): In this window In a new window Download PPT slide   Figure 8. Siw14 does not affect the phosphorylation state of Slt2. Exponentially growing wild-type (wt; W303a), whi2, siw14, and slt2siw9 strains, with or without a multicopy, pSIW14 as indicated, were incubated in YEPD for 1 hr in the presence or absence of 6 mM caffeine. Total protein extracts were prepared and probed by Western blotting using polyclonal anti-active Slt2 antisera. The loading control is a nonspecific band that does not alter its intensity upon caffeine treatment.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Whi2 cells are defective in the organization of the actin cytoskeleton and endocytosis:
We have shown here that the effect of a whi2 allele is pleiotropic. In addition to the failure to respond to nutrient limitation by ceasing cell division, whi2 cells are sensitive to a wide range of stresses, have a disorganized actin cytoskeleton upon nutrient limitation, and are defective in fluid-phase endocytosis. Overexpression of WHI2 has been reported to rescue the stress sensitivity of an rsp5 mutation (KAIDA et al. 2002 ). Rsp5 is a ubiquitin protein ligase that is thought to regulate the endocytic machinery and is required for normal stress response (reviewed in ROTIN et al. 2000 ). It is responsible for the mono-ubiquitination and consequent internalization of many membrane proteins such as permeases, transporters, and receptors, including the Ste2 -factor receptor. Rsp5 interacts with Pan1, a protein that links the endocytic machinery to the plasma membrane (ZOLADEK et al. 1997 ), and its location to the plasma membrane and perivacuolar structures is Sla2 dependent (WANG et al. 2001 ). The suppression of the rsp5 phenotype by WHI2 overexpression suggests that Whi2 may be a positive regulator of endocytosis, which would be consistent with the defects in endocytosis reported here in a whi2 mutant.

A synthetic lethal screen isolated further mutants with the same pleiotropic defects as whi2:
Using a colony-sectoring assay, we isolated a number of other mutants that had some or all of the phenotypes of a whi2 mutant. Of these prs3, fen2, and siw14 were particularly interesting since they showed sensitivity to stresses such as caffeine and 1 M NaCl and failed to respond by halting cell proliferation in response to nutrient depletion.

The amino acid sequence of Siw14 suggests that it is a member of a small family of tyrosine phosphatases. There are two homologs of SIW14 in the yeast genome: OCA1 (33% identity, 51% similarity) and YNL056w (35% identity and 58% similarity). Oca1 is required for cell cycle arrest in response to acid linoleic hydroperoxide, a lipid peroxidation product that accumulates during oxygen stress (ALIC et al. 2001 ), so other members of this family are also concerned with stress response. We mutated the active site of Siw14 and found that catalytically dead alleles still complement the caffeine sensitivity of the siw14 mutation. Clearly, Siw14 has a function that is not dependent on tyrosine phosphatase activity. However, it is likely that Siw14 does have tyrosine phosphatase activity, because the catalytically dead mutants are unable to suppress the caffeine sensitivity of the slt2^siw9 allele or to enhance the sensitivity of the slt2 allele. Thus, Siw14 appears to be a bifunctional protein.

The inability of slt2 mutants to arrest upon nutrient deprivation and an abnormal distribution of actin in slt2 cells has been reported previously (MAZZONI et al. 1993 ; COSTIGAN and SNYDER 1994 ). Null alleles of SIW14 also show cytoskeletal defects. When depleted of nutrients, siw14 cells contain a single large clump of actin and associated proteins such as Abp1, Cof1, and Sac6. Furthermore, the septin cytoskeleton shows a similar phenotype, as Cdc11 accumulates in large bars. This phenotype is strikingly similar to that of an ark1 prk1 mutant. Ark1 and Prk1 are two protein kinases that regulate the actin cytoskeleton (COPE et al. 1999 ; ZENG and CAI 1999 ; ZENG et al. 2001 ). In an ark1 prk1 double mutant, actin was found in large clumps, similar to the appearance of actin clumps that we observe in stationary-phase whi2 and siw14 cells (COPE et al. 1999 ). Overexpression of either Prk1 or Ark1 results in a different type of actin abnormality: the formation of large cytoplasmic bars. We showed that overexpression of SIW14 in a prk1 results in an apparent loss of all forms of filamentous actin and is lethal. The similar actin cytoskeletal defects in both siw14 and ark1 prk1 mutants as well as the genetic interaction between prk1 and SIW14 overexpression suggest that SIW14 may influence budding during nutrient deprivation through the actin cytoskeleton.

The actin cytoskeleton participates in endocytosis, and siw14 mutants are defective in fluid-phase endocytosis. Siw14 has been shown to physically interact with Ypt53, a Rab5-like GTPase required for vesicle-mediated transport (UETZ et al. 2000 ). YPT53 shares homology with YPT52 and VPS21 (YPT51). It is thought that Vps21, Ypt52, and Ypt53 are required for the early steps in endocytosis (SINGER-KRUGER et al. 1994 ; PRESCIANOTTO-BASCHONG and RIEZMAN 2002 ). A null allele of VPS21 causes an impairment of endocytosis that is more severe when combined with ypt52 and ypt53 alleles. We found that, like siw14 mutants, vps21 and ypt52 mutants upon nutrient limitation show a failure to arrest cells and have a disorganized actin cytoskeleton. We also found that vps21 and ypt52 mutants are sensitive to caffeine and 1 M NaCl and display actin clumps in stationary phase. We also examined a ypt53 strain but failed to see any phenotypic abnormalities. This may be because the function of Ypt53 is redundant with Vps21 and Ypt52. However, taken together, these observations support the conclusion that a normal response to nutrient and other stresses involves actin organization and endocytosis.

FEN2 was first identified in a search for mutants resistant to fenproprimorph, an inhibitor of ergosterol biosynthesis (MARCIREAU et al. 1996 ). Cells harboring a fen2 mutation were shown to have a threefold decrease in ergosterol levels. Subsequently, it was shown that Fen2 is a membrane pantothenate transporter (STOLZ and SAUER 1999 ). Pantothenate is essential for the biosynthesis of coenzyme A, which is a carrier of activated C₂ units in sterol biosynthesis. Thus, the reduction in ergosterol levels in a fen2 mutant is due to reduced availability of pantothenate. Ergosterol is required for the proper fluidity and function of cell membranes and for endocytosis (HONGAY et al. 2002 ). Thus, the reduction in endocytosis in a fen2 mutant that we observed can be satisfactorily explained by the known function of Fen2.

We isolated a thr4 mutation in the synthetic lethal screen and we found that a thr4 mutant was sensitive to 1 M NaCl and was defective in endocytosis. The involvement of threonine synthetase was initially puzzling. However, a thr4 mutation was recovered in a screen for mutations that are colethal with sec13, which is defective in the transport of the general amino acid permease from the Golgi to the cell surface (ROBERG et al. 1997 ). Thus, it is possible that Thr4 does have an unexpected role in protein trafficking.

Mutations known to affect the organization of the actin cytoskeleton and reduce endocytosis decouple growth and cell division:
Although the molecular functions of the Whi2, Siw14, Fen2, and other Siw genes are diverse, they share the common property that mutations result in a disorganized actin cytoskeleton and/or reduce endocytosis. This observation led us to examine the phenotype of other mutants known to be defective in the organization of the actin cytoskeleton and endocytosis. Many mutants defective in endocytosis did not show any obvious deficiencies in response to nutrient stress. However, we found that rvs161, sla1, sla2, vrp1, vps21, ypt52, ypt53, and end3 had the same phenotype as the original whi2 mutation. The phenotypes of vrp1 and rvs161 were particularly strong, resulting in a marked reduction in cell size compared to wild-type cells. It is not clear why only some mutants studied here show a reduction in cell size. However, one important point to be considered is that we have measured cell volume, which includes both the cytoplasmic and vacuolar compartments. Many of the mutants clearly result in enlarged vacuoles, which could be masking a reduction in the size of the cytoplasmic compartment. It is also important to note that the sizes measured here refer to stationary-phase cells. We observed no size reduction in exponentially growing cells.

Recently, two systematic surveys have yeast deletion sets to identify genes that regulate cell size in S. cerevisiae. The work of JORGENSEN et al. 2002 focused on exponentially growing cells and thus is not directly comparable to the experiments reported here. ZHANG et al. 2002 initially screened the deletion collection for mutants that had an abnormally small cell size in stationary-phase cultures and then tested for size abnormalities in exponentially growing cells. This work identified whi2 as one of the 20 mutations that caused abnormally small size in stationary phase. None of the other mutations identified here that cause a reduction in cell size were present in the list of ZHANG et al. 2002 . However, FEN2 (YCR028c) was listed in a table of mitochondrial mutants that showed a reduction in cell size. The criterion for mitochondrial mutations was a failure to grow on glycerol. As discussed above, the molecular defect has been identified in fen2 and it seems likely that it is misclassified as a mitochondrial mutant. VPR1 was also present in a list of genes whose deletion resulted in small cell size, but that for various reasons failed a quality control test. The rvs161 allele also showed a size reduction, but was also omitted for quality control reasons (B. SCHNEIDER, personal communication). Thus, the results presented here are broadly consistent with the results of the systematic gene deletion survey.

The proteins that are required for the proper nutrient response function either in the first steps of endocytosis or in the actin complex that mediates endocytosis. Taken together, these observations suggest that the actin/endocytosis complex is required for cells to cease cell division in response to nutrient stress. We suggest two possible explanations. First, endocytosis could result in the internalization of membrane proteins required for growth and budding. Second, fluid-phase endocytosis could be required for the uptake of small molecules from the medium used to sense cell density. Such molecules may accumulate to form the signal to cease cell division in the high cell densities found in stationary-phase culture. A precedent for this may be the way that Candida albicans cells undergo only the yeast-to-hypha transition at low cell densities. It has recently been shown that the accumulation of farnesol provides the signal that is used by C. albicans cells to sense cell density (HORNBY et al. 2001 ; OH et al. 2001 ).

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Cambridge Antibody Technology, Cambridge CB1 6GH, United Kingdom.
³ Present address: Oxford Biomedica, Oxford OX4 4GA, United Kingdom.
⁴ Present address: Dipartimento di Biotechnologie Agrarie, Ed Ambientali Universita' Di Ancona, Ancona 60100, Italy.

	ACKNOWLEDGMENTS

We thank Mike Snyder, Kevin Costigan, Kathryn Ayscough, Mike Tyers, and Giovanna Lucchini for strains and plasmids and Kathryn Ayscough for critical reading of the manuscript. This work was supported by a project grant from the Biotechnology and Biological Sciences Research Council (BBSRC). A.C. received a BBSRC Cooperative Award in Science and Engineering studentship. K.B. and P.R. were supported by BBSRC research training studentships.

Manuscript received July 28, 2003; Accepted for publication October 27, 2003.

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