four novel suppressors of gic1 gic2 and their roles in ... · mlf3 fusions were expressed under the...

Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.058180

Four Novel Suppressors of gic1 gic2 and Their Roles in Cytokinesis andPolarized Cell Growth in Saccharomyces cerevisiae

Meghal Gandhi,*,1 Bruce L. Goode† and Clarence S. M. Chan*

*Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712 and†Department of Biology and Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02454

Manuscript received March 14, 2006Accepted for publication June 23, 2006

ABSTRACT

Gic1 and Gic2 are two Cdc42/Rac interactive binding (CRIB) domain-containing effectors of Cdc42-GTPase that promote polarized cell growth in S. cerevisiae. To identify novel genes that functionallyinteract with Gic1 and Gic2, we screened for high-copy suppressors of a gic1 gic2 temperature-sensitivestrain. We identified two pairs of structurally related genes, SKG6-TOS2 and VHS2-MLF3. These genes havebeen implicated in polarized cell growth, but their functions have not previously been characterized. Wefound that overproduction of Skg6 and Tos2 in wild-type cells causes aberrant localization of Cdc3 septinand actin structures as well as defective recruitment of Hof1 and impaired formation of the septum at themother-bud neck. These data suggest a negative regulatory function for Skg6 and Tos2 in cytokinesis.Consistent with this model, deletion of SKG6 suppresses the growth defects associated with loss of HOF1, apositive regulator of cytokinesis. Our analysis of the second pair of gic1 gic2 suppressors, VHS2 and MLF3,suggests that they regulate polarization of the actin cytoskeleton and cell growth and function in apathway distinct from and parallel to GIC1 and GIC2.

THE establishment of cell polarity is required fordifferentiation in most cell types and is a critical

step in cellular morphogenesis (Drubin and Nelson

1996; Johnson 1999; Nelson 2003). Budding inSaccharomyces cerevisiae involves polarized cell growthduring which the actin cytoskeleton is asymmetricallyorganized. This process is both spatially and temporallyregulated to synchronize with progression of the cellcycle. Bud emergence is accompanied by entry of cellsinto a new cell cycle (G1-to-S transition) and establish-ment of the mother–daughter axis of polarization.F-actin structures including actin cables and corticalactin patches are directed to the growing bud cortex topromote apical bud growth until G2–M phase whenthey are distributed in the daughter cell to promoteisotropic bud growth. Upon sufficient bud growth,actin cables and patches are briefly depolarized in boththe mother and the daughter cell until cell cycle cuessignal their repolarization toward the mother-bud neckto promote cytokinesis (Pruyne and Bretscher 2000b).

Cytokinesis in budding yeast is accomplished byconcerted actions of actomyosin ring closure andseptum formation at the site of cell division (Bi et al.1998). Evolutionarily conserved structural GTPases,called septins, form a system of filaments and are

essential for cytokinesis (Moffat and Andrews 2003).Early in the cell cycle, septins mark the site for futurecell division, which coincides with the site of bud emer-gence (Longtine and Bi 2003). Subsequently, septinspromote cytokinesis by recruiting proteins to the budneck required for formation of the actomyosin ring(e.g., Myo1, F-actin, and Cyk1) and the primary septum(e.g., chitin synthase II components) (Schmidt et al.2002). Septins also are required for reorientation ofactin cables to the bud neck at cytokinesis, which targetsvesicle delivery for septum formation at the divisionplane (Adams and Pringle 1984).

Hof1 (also known as Cyk2) is a homolog of Schizo-saccharomyces pombe Cdc15, which is a founding memberof the PCH/FCH family of conserved proteins involvedin actin-based processes and shown to organize sterol-rich membrane domains at the site of cell division(Lippincott and Li 2000; Takeda et al. 2004). Hof1plays an important role in cytokinesis in S. cerevisiae,localizing as a ring structure to the mother-bud neckand stabilizing the actomyosin ring during contraction(Lippincott and Li 1998). However, on the basis of ge-netic evidence the primary cytokinetic function of Hof1appears to be in septum formation (Vallen et al. 2000).Cells lacking Hof1 display cytokinesis defects appearingas chains of cells with connected cytoplasms. This phe-notype of hof1 cells results from failure in septum for-mation between two cell bodies before subsequentrounds of budding (Lippincott and Li 1998; Vallen

et al. 2000)

1Corresponding author: Rosenstiel Basic Medical Science ResearchCenter, Brandeis University, 415 South St., Waltham, MA 02454.E-mail: [email protected]

Genetics 174: 665–678 (October 2006)

Polarized growth is accompanied not only by reorga-nization of the actin cytoskeleton, but also by insertionof a new membrane at the cell cortex, biosynthesis ofcell-wall material at the sites of active growth, andremodeling of the cell wall required for cell expansion.Both actin cytoskeleton reorganization and cell-wallremodeling are controlled by Rho-type GTPases. Theevolutionarily conserved GTPase Cdc42 is recruited tothe incipient bud site and (through activation of itsdownstream effectors) directs polarized redistributionof the actin cytoskeleton (Ziman et al. 1993). Actin ca-bles serve as tracks for the myosin V-dependent deliveryof secretory vesicles containing new plasma mem-brane and cell-wall material to sites of polarized growth(Pruyne and Bretscher 2000a). Actin patches, in addi-tion to being sites of dynamic actin assembly and endo-cytosis (Pruyne and Bretscher 2000a), provide siteswhere deposition of new cell-wall material occurs(Mulholland et al. 1994). In contrast to Cdc42, Rho1and Rho2 affect cell-wall synthesis, both by directlystimulating the glucan synthase complex and by in-directly increasing expression of cell-wall biosynthesismachinery through upregulation of the Pkc1-MAPkinase pathway (Cabib et al. 1998; Schmidt and Hall

1998).Gic1 and Gic2 are two related Cdc42/Rac-interactive

binding domain-containing effectors of Cdc42 firstidentified as multicopy suppressors of a BEM2 (Rho-type GTPase) mutant allele and shown to bind anactivated form of Cdc42 (Brown et al. 1997; Chen

et al. 1997). Deletion of both GIC1 and GIC2 results inloss of cell polarity and cell growth at elevated temper-atures. gic1 gic2 cells are enlarged and abnormallyrounded with defects in bud-site selection and actinpolarization (Chen et al. 1997). However, the mecha-nism by which Gic1 and Gic2 contribute to polarizedcell growth remains unclear. To gain new insights, weperformed a high-copy suppression screen on a gic1 gic2strain and identified two pairs of structurally relatedgenes that function with GIC1 and GIC2 in directingpolarized growth and cell division.

MATERIALS AND METHODS

Strains, media, genetic techniques, and growth conditions:Yeast strains used in this study are listed in Table 1. Preparationof rich medium (YEPD), synthetic minimal medium (SD), andSD with necessary supplements was performed as described(Rose et al. 1990). Standard methods of yeast genetics andrecombinant DNA manipulations were carried out (Sambrook

et al. 1989; Guthrie and Fink 1991). Construction of genedeletion mutants and replacement of the chromosomalnative promoters by the inducible GAL1 promoter werecarried out by a one-step PCR-mediated homologous recom-bination technique (Longtine et al. 1998). For overexpres-sion of genes under the control of the GAL1 promoter,cultures were grown at 26� in rich medium containingraffinose (2%) to a cell density of 1 3 107 cells/ml, followedby dilution to 2.5 3 106 cells/ml with rich medium containing

galactose (4%) and incubation at 26� or 37� for indicatedperiods of time. Strains expressing chromosomally taggedHOF1-GFP and MYO1-GFP fusions were generated by trans-forming yeast strains with the YCplac111-based integrationplasmids pSK1051 and pSK1052 (gift from K. S. Lee, NationalCancer Institute, Bethesda, MD) bearing HOF1-GFPTLEU2and MYO1-GFPTLEU2 fragments, respectively.

Cloning of high-copy suppressors of gic1 gic2: Briefly, atemperature-sensitive (Ts�) gic1-D1TLEU2 gic2-1THIS3 ura3-52 strain (CCY1024-19C) that fails to grow at $33� wastransformed with a yeast genomic DNA library constructedin the high-copy number URA3-plasmid YEp24 (Carlson andBotstein 1982). Ura1 transformants were selected on supple-mented SD agar lacking uracil. After 20–24 hr of growth at 26�,the plates containing Ura1 transformants were shifted to 35�.Three days later, viable Ura1 transformants were identified.Approximately 100,000 library transformants were screened,which yielded 426 transformants able to grow at 35�. Plasmidsfrom these transformants were recovered and amplified inEscherichia coli. Colony hybridization of E. coli strains bearingeach of these plasmids with genes that were known to suppressthe temperature-sensitive growth defect of gic1 gic2 cellsrevealed that 232 of the suppressor plasmids contained pre-viously identified suppressor genes (including BEM1, CDC42,GIC1, GIC2, MSB3, and SSD1-v1) (Brown et al. 1997; Chen

et al. 1997; Bi et al. 2000). Restriction enzyme digestion andpartial sequencing of the remaining 194 candidates revealedthat they represented 11 unique classes of plasmids (Figure1A). Comparison of the insert sequences from different classesof plasmid against the Saccharomyces Genome Database en-abled prediction of a single ORF on each plasmid that likelywas responsible for suppression of the temperature-sensitivegrowth defect of gic1 gic2 cells. This was confirmed bysubcloning individual ORFs where possible or by disrupting/deleting that ORF from the library plasmid and testing theability of modified plasmids to suppress the growth defects ofgic1 gic2 cells at restrictive temperatures.

Cytological techniques: Microscopy was performed usingZeiss Axioscope (Carl Zeiss, Oberkochen, Germany). Imageswere captured using a MicroMax CCD camera (PrincetonInstruments, Trenton, NJ) and processed using IPLab spec-trum (Scanalytics, Fairfax, VA) software.

For examining the morphological features, cells were fixedwith formaldehyde (3.7%; EM Sciences, Gibbstown, NJ) for0.5–1 hr at room temperature and washed twice with PBSbuffer. Actin staining was carried out essentially as describedpreviously (Pringle et al. 1989). For staining nuclei, fixed cellswere resuspended in PBS buffer containing 1 mg/ml 49,6-diamidino-2-phenylindole (DAPI; Sigma Chemical, St. Louis)and incubated for 10 min before visualization. Chitin at thebud neck was examined by staining fixed cells with 0.01 mg/mlcalcofluor (Sigma Chemical). To evaluate cell separation,fixed cells were washed twice with PBS, once with sorbitolbuffer (1 m sorbitol in 50 mm K-phosphate, pH 7.5), andincubated with 0.2 mg/ml zymolyase 20T (ICN Biomedicals,Aurora, OH) in sorbitol buffer containing 2 mm DTTat 37� for10 min. More than 90% of such treated cells lost their refractileappearance as observed by light microscopy, indicating cell-wall removal was efficient.

For experiments to detect the subcellular localization ofCdc3-GFP, Myo1-GFP, and Hof1-GFP, cells were cultured at 37�for indicated periods of time, fixed with 1% formaldehyde onice for 10 min, washed once with ice-cold PBS buffer, andscored immediately thereafter. To detect the subcellularlocalization pattern of Vhs2 and Mlf3, GFP-VHS2 and GFP-MLF3 fusions were expressed under the ACT1 promoter froma low-copy plasmid pTD125 and visualized in live cells fromlogarithmically growing cultures.

666 M. Gandhi, B. L. Goode and C. S. M. Chan

RESULTS

Identification of high-copy suppressors of gic1 gic2cells: gic1 gic2 cells are viable but exhibit a Ts� growthdefect at $33� (Chen et al. 1997). We used this phe-notype to isolate genes, which upon overexpressionfrom high-copy number plasmids could alleviate the Ts�

growth defect of gic1 gic2 cells. With this approach, weanticipated the identification of genes that functionallyinteract with GIC1 and GIC2 either in a common linearpathway or in redundant parallel pathways to facilitatepolarized cell growth. We identified as suppressors manyknown polarity-related genes, including AXL2, BNI1,CLN2, MSB1, MSB2, RSR1, and STE20 (lacking auto-inhibitory sequences upstream of nucleotide 355 of theSTE20 ORF, and hence ND-118-STE20) (Figure 1A).Surprisingly, overexpression of SSN6, which encodes aconserved transcriptional repressor that controls theexpression of �3% of yeast genes (Smith and Johnson

2000), also complemented the Ts� growth defect of gic1

gic2 cells. Since Ssn6 functions in a complex with theTup1 transcriptional repressor (Keleher et al. 1992), weexamined whether overexpression of TUP1 also couldcomplement the Ts� growth defect of gic1 gic2 cells.Indeed, a 2m plasmid bearing TUP1 suppressed the Ts�

defect of gic1 gic2 cells (Figure 1A), albeit slightly lessefficiently than the SSN6 plasmid.

Interestingly, three ORFs identified from our screenhad no well-characterized function in polarized cellgrowth at the time that we identified them (see belowfor recent reports implicating these genes in polaritypathways). These ORFs were YHR149c (SKG6), YGR221c(TOS2), and YIL135c (VHS2) (Figure 1A). Skg6 and Tos2share sequence homology with each other, and Vhs2is homologous in sequence to another S. cerevisiae pro-tein, Mlf3. Therefore we tested whether overexpressionof MLF3 could suppress the Ts� phenotype of gic1 gic2cells. Indeed, a 2m plasmid bearing MLF3 partially sup-pressed the Ts� defect of gic1 gic2 cells (Figure 1A). Notethat simultaneous overexpression of these homologous

TABLE 1

Yeast strains used in this study

Strain Genotype

CCY1024-19C a his3-D200 leu2-3,112 trp1-1 ura3-52 gic1-D1TLEU2 gic2-1THIS3CCY1137-3B a ade2 his3-D200 leu2-3,112 ura3-52 trp1-1 hof1-D101TspHIS5 with pCC1368 (CEN, URA3, HOF1)CCY1176-11A a lys2-801 his3-D200 leu2-3,112 ura3-52 trp1-1 skg6-D1Tkan tos2-D2TURA3CCY1244-4D a his3-D200 leu2-3,112 trp1-1 ura3-52 hof1-D101TspHIS5CCY1244-7C a his3-D200 leu2-3,112 trp1-1 ura3-52 hof1-D101TspHIS5 skg6-D1TkanCCY1244-10D a his3-D200 leu2-3,112 trp1-1 ura3-52 hof1-D101TspHIS5 tos2-D2TURA3CCY1244-17C a his3-D200 leu2-3,112 trp1-1 ura3-52 hof1-D101TspHIS5 skg6-D1Tkan tos2-D2TURA3CCY1292-10C a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 vhs2-D1TTRP1 mlf3-D1TkanCCY1292-1A a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52CCY1292-1D a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 mlf3-D1TkanCCY1292-4B a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 vhs2-D1TTRP1CCY1292-5B a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 vhs2-D1TTRP1 mlf3-D1TkanCCY1338 a/a ade2/1 lys2-801/1 his3-D200/his3-D200 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-52/ura3-52 vhs2-D1T

TRP1/ vhs2-D1TTRP1 mlf3-D1Tkan/mlf3-D1TkanCCY1708-1A a ade2 his3-D200 leu2-3,112 lys2-801 ura3-52 spHIS5-pGAL1-3HA-SKG6CCY1708-1A-1 a ade2 his3-D200 leu2-3,112 lys2-801 ura3-52 spHIS5-pGAL1-3HA-SKG6 MYO1-GFPTLEU2CCY1708-1A-2 a ade2 his3-D200 leu2-3,112 lys2-801 ura3-52 spHIS5-pGAL1-3HA-SKG6 HOF1-GFPTLEU2CCY1709-2B a his3-D200 leu2-3,112 lys2-801 ura3-52 spHIS5-pGAL1-3HA-TOS2CCY1710-3C a ade2 his3-D200 leu2-3,112 lys2-801 ura3-52CCY1710-3C-1 a ade2 his3-D200 leu2-3,112 lys2-801 ura3-52 MYO1-GFPTLEU2CCY1710-3C-2 a ade2 his3-D200 leu2-3,112 lys2-801 ura3-52 HOF1-GFPTLEU2CCY1720 a/a ade2/1 lys2-801/1 his3-D200/his3-D200 leu2-3,112/leu2-3,112 trp1-1/trp1-1

ura3-52/ura3-52 vhs2-D1TTRP1/vhs2-D1TTRP1CCY1721 a/a ade2/1 lys2-801/1 his3-D200/his3-D200 leu2-3,112/leu2-3,112 trp1-1/trp1-1

ura3-52/ura3-52 mlf3-D1Tkan/mlf3-D1TkanCCY1726-18C a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 gic1-D1TLEU2 gic2-1THIS3CCY1726-2B a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 gic1-D1TLEU2 gic2-1THIS3 vhs2-D1TTRP1CCY1726-4D a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 gic1-D1TLEU2 gic2-1THIS3 vhs2-D1TTRP1 mlf3-D1TkanCCY1726-5B a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52 gic1-D1TLEU2 gic2-1THIS3 mlf3-D1TkanDBY1829 a lys2-801 his3-D200 leu2-3,112 trp1-1 ura3-52DBY1830 a/a ade2/1 lys2-801/1 his3-D200/his3-D200 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-52/ura3-52

Strains DBY1829 and DBY1830 are from David Botstein’s laboratory. Strain 1024-19C is from Chen et al. (1997). All other strainswere constructed specifically for this study.

Skg6, Tos2, Vhs2 and Mlf3 in Cell Polarity 667

gene pairs, SKG6 and TOS2, or VHS2 and MLF3, showedno additive effects in suppressing the gic1 gic2 phe-notype (not shown).

We next examined the ability of these suppressors torescue actin organization defects of gic1 gic2 cells. Mostsuppressor plasmids decreased the fraction of gic1 gic2cells with a depolarized actin cytoskeleton, reflected byan overall decrease in the fraction of unbudded cells(Figure 1B). The ability of these suppressors to rescueactin polarization defects of gic1 gic2 cells correlatedwith their ability to suppress the Ts� growth defect(Figure 1A), suggesting that these phenotypes may beinterdependent.

Since SKG6, TOS2, VHS2, and MLF3 suppressed theTs� growth and actin organization defects of gic1 gic2cells, we further investigated their roles in polarized cellgrowth.

Skg6 and Tos2 are not essential for polarized cellgrowth: Skg6 and Tos2 proteins are 734 and 622 aminoacids in length, respectively. Their primary sequencesshare 34% identity and 47% similarity (supplementalFigure 1 at http://www.genetics.org/supplemental/).Each is predicted to contain a single transmembranedomain within the N-terminal region (residues 71–99 inSkg6 and residues 37–64 in Tos2), and Skg6 containstwo putative peptide cleavage sites (after residues 23 and95). Both Skg6 and Tos2 localize to sites of polarizedgrowth (i.e., throughout the bud cortex in small-buddedcells, as a cap along the bud cortex in medium-budded

cells and as a double-ring structure at the bud neck inlarge-budded cells; Drees et al. 2001 and our unpub-lished results). Skg6 is known to interact in the two-hybrid assay with the Cdc42 effector Cla4 (Uetz et al.2000) and two potential negative regulators of Cdc42,Zds1, and Zds2 (Bi and Pringle 1996), which them-selves interact in the two-hybrid assay with Gic1 and Gic2(Uetz et al. 2000; Drees et al. 2001). Tos2 is known tointeract in the two-hybrid assay with Pkc1 and Cdc24,the guanine nucleotide exchange factor for Cdc42(Drees et al. 2001). In fact, one study indicated thatTos2 may help anchor Cdc24 to sites of polarized growth(Toenjes et al. 2004). These observations, together withour identification of SKG6 and TOS2 as high-copy sup-pressors of gic1 gic2, suggest that these two genes likelyregulate polarized cell growth.

To investigate further the potential function of SKG6and TOS2 in polarized growth, we generated strains thatlack one or both genes. skg6 and tos2 single-mutant cellsand skg6 tos2 double-mutant cells each were viable andshowed normal rates of growth at a range of temper-atures from 13� to 37�. Mutant cells also displayednormal cell size and morphology and had no obviousdefects in bud-site selection, actin cytoskeleton organi-zation, mating projection formation, or sporulation(not shown).

Skg6 or Tos2 overproduction leads to defects inapical-to-isotropic growth switch and cytokinesis: Wenext tested whether overproduction of Skg6 or Tos2

Figure 1.—(A) Suppression of the Ts� growthdefect of gic1 gic2 cells by high-copy suppressorplasmids. Suspensions of haploid gic1-D1TLEU2gic2-1THIS3 cells (CCY1024-19C) carrying differ-ent 2m-based plasmids were spotted on YEPDagar and allowed to grow at the indicated temper-atures for 2 days. The plasmids used were pRS426(empty vector control), pCC1290 (RSR1),pCC1291 (AXL2), pCC1284 (MSB2), pPB191(MSB1), YEp352-BNI1 (BNI1), pCC1294(ND118-STE20), pCC1295 (CLN2), pCC1293(SSN6), pAJ181 (TUP1, gift from A. Johnson),pCC1478 (SKG6), pCC1574 (TOS2), pCC1606(VHS2), pCC1635 (MLF3), and pCC904 (GIC1,positive control). Note that TUP1 and MLF3 werenot identified in the original screen. (B) Suppres-sion of the actin polarization defect of gic1 gic2cells by high-copy suppressor plasmids. Cellsshown in A were incubated at 33� for 4 hr in sup-plemented SD medium lacking uracil to a densityof �1 3 107 cells/ml, fixed, stained with rhoda-mine–phalloidin, and examined by fluorescentmicroscopy. For each sample, �200 cells werescored. Shown is the percentage of cells thatappeared unbudded, including those with de-polarized (open) or polarized (solid) actinorganization.


perturbs polarized growth in wild-type cells. To this end,we replaced the chromosomal native promoter of SKG6and TOS2—either singly or in combination—with aninducible GAL1 promoter. Cells were grown in richmedium containing galactose to induce the overpro-duction of Skg6 or Tos2 at 26�. This resulted in theappearance of a small fraction of cells with abnormallyelongated buds, an effect that was greatly exacerbatedat 37� (Figure 2A and Table 2). Furthermore, simulta-neous overproduction of Skg6 and Tos2 increased thefraction of cells with elongated buds. Interestingly, asignificant fraction of Skg6-overproducing cells (and, toa lesser extent, Tos2-overproducing cells) had a secondbud that in most instances emerged from the previouslyformed elongated bud (denoted by arrowheads in

Figure 2A). The appearance of multibudded cells wasaccompanied by a reduction in the fraction of cells thatwere unbudded or had a single bud. Together, thesephenotypic changes suggested that overproduction ofSkg6 or Tos2 disrupts the switch from apical-to-isotropicgrowth and causes a delay or defect in the normalexecution of cytokinesis. To ascertain whether theelongated/multibudded phenotype of Skg6- and Tos2-overproducing cells results from a defect in cell divisionor, alternatively, from a defect in DNA duplication andsegregation as seen for the Ts� cdc4 mutant cells (Goh

and Surana 1999), we examined their nuclei by DAPIstaining. Like wild-type cells, Skg6- and Tos2-overpro-ducing cells showed normal nuclear division andsegregation regardless of the shape and number of

Figure 2.—Overproduc-tion of Skg6 or Tos2 resultsin cytokinesis and septinorganization defects. (A)Morphology of haploidwild-type cells (CCY1710-3C) and cells overproducingHA–Skg6 (CCY1708-1A) orHA-Tos2 (CCY1709-2B) un-der the control of the GAL1promoter for 6 hr at 37�and then fixed in formalde-hyde. Cells with ‘‘bud froma bud’’ morphology aremarked with arrowheads.(B) DAPI stainingof cells in A to examineDNA distribution. HA-Tos2-overproducing cells (notshown) exhibited DNAdistribution similar to HA-Skg6-overproducing cells.(C) Morphology of wild-type and HA-Skg6-over-

producing cells from A before and after zymolyase treatment. Note that the majority of cells in both strains lost their refractileappearance (indicative of complete cell-wall digestion) but the multibudded phenotype of HA–Skg6-overproducing cells per-sisted, confirming the cytokinesis defect. (D) Localization of Cdc3-GFP in wild-type cells (CCY1710-3C containing pRS316-CDC3-GFP, a gift from M. Longtine) and in cells overproducing HA-Skg6 (CCY1708-1A containing pRS316-CDC3-GFP) for6 hr at 37�. All cells are shown at the same magnification.

TABLE 2

Morphological analysis of cells overproducing Skg6 or Tos2

Budded (%)

Growthcondition

Proteinoverproduced

Unbudded(%)

Single regularbud

Single elongatedbud

Two separatebuds

‘‘Bud from abud’’

26� — 46 54 0 0 0YEP-raffinose HA-Skg6 40 60 0 0 0

HA-Tos2 45 55 0 0 0

26� — 49 51 0 0 0YEP-galactose for 6 hr HA-Skg6 41 56 3 0 0

HA-Tos2 50 48 2 0 0

37� — 46 51 0 3 0YEP-galactose for 6 hr HA-Skg6 34 14 27 6 19

HA-Tos2 46 38 10 3 3


their buds (Figure 2B). Additionally, the multibuddedphenotype of Skg6- or Tos2-overproducing cells couldnot be resolved by zymolyase treatment (Figure 2C),further suggesting that the defect lies in cytokinesisrather than cell separation.

Skg6 or Tos2 overproduction causes septin misloc-alization: Current models for cytokinesis in S. cerevisiaesuggest that there are two parallel pathways—one in-volving assembly and contraction of an actomyosin ringand the other involving formation of a septum—both ofwhich are spatially and temporally coordinated. In theabsence of actomyosin ring formation, the septumpathway is sufficient to drive cell division. Key compo-nents of each of these pathways, Myo1 and Hof1, re-spectively, localize independently of each other at thebud neck. However, both proteins depend on septins fortheir recruitment and maintenance at the bud neck,making septins essential for cytokinesis (Field andKellogg 1999; Bi 2001).

The cytokinesis defect in Skg6- and Tos2-overproduc-ing cells prompted us to examine the localization ofseptins in these cells. CDC3 is an essential gene encod-ing one of the septin proteins, and cdc3 conditionalmutants arrest with hyperelongated buds (Kim et al.1991; Longtine and Bi 2003). In budded wild-type cells,we found that Cdc3-GFP localizes as an hourglassstructure at the bud neck (Figure 2D, a and b), oftenappearing as two closely apposed rings as previouslydescribed (Kim et al. 1991). However, in elongated/multibudded cells overproducing Skg6 or Tos2, Cdc3-GFP was aberrantly organized and/or mislocalized (72and 20%, respectively; Figure 2D). In many cells, Cdc3-GFP was found at some distance from the bud neck,skewed toward either the daughter (Figure 2D, g) or themother (not shown). In other cells, Cdc3-GFP appearedas small dots or short bars that were mislocalized againstone side of the bud (Figure 2D, e, h, and j). Occasion-ally, the two rings of Cdc3-GFP were split asymmetrically(Figure 2D, f) or separated excessively (Figure 2D, c).Since localization of different septin proteins is in-terdependent (Haarer and Pringle 1987; Kim et al.1991), it is likely that localization of the other septins isalso impaired in Skg6- and Tos2-overproducing cells.

Localization of Myo1, F-actin, and Hof1 in Skg6-overproducing cells: The results above suggest thatoverproduction of Skg6 (and Tos2) interferes with thelocalization of the cell division machinery and therebywith the normal execution of cytokinesis. To furtherdissect which of the aforementioned cytokinesis path-ways is likely impaired, we examined in Skg6-over-producing cells the localization patterns of severalcytokinetic components known to be downstream of,and dependent on, septins: Myo1, F-actin, and Hof1.

Myo1–GFP expressed from its chromosomal locusunder the endogenous promoter was found at themother-bud neck of medium- to large-budded cells inboth wild-type cells and Skg6-overproducing cells (74 vs.

61%, respectively; n¼ 100 cells; Figure 3A). Further, thefraction of cells showing Myo1-GFP as a dot (indicating acontracted actomyosin ring) was similar for wild-typeand Skg6-overproducing cells. However, 64% of elon-gated/multibudded Skg6-overproducing cells showed aMyo1–GFP band that was off center from the mother-bud junction (Figure 3A, c), skewed toward either themother or the daughter cell (Figure 3A, e and f,respectively), a pattern never observed in wild-type cells.Thus, while overexpression of Skg6 does not impairrecruitment of Myo1 to the bud neck, it affects Myo1spatial organization. It is possible that these defects inMyo1 arise as a consequence of the defects in septinorganization and localization caused by Skg6 overex-pression (above), especially given that Myo1 localizationdepends on septins.

Next, we examined the effects of Skg6 overproduc-tion on polarized actin organization and Hof1-GFPlocalization at the bud neck, both of which depend onseptins and are necessary for septum formation and cellseparation (Adams and Pringle 1984; Lippincott andLi 1998; Vallen et al. 2000). Approximately 72% ofSkg6-overproducing cells with elongated buds showedactin repolarization to the bud neck that precedescytokinesis. However, unlike the condensed double ringof actin patches seen in wild-type cells, .74% of thesecells demonstrated a fanned-out actin patch organiza-tion that was often biased toward one side of the mother-bud neck (Figure 3B and Table 3). Analysis of Hof1-GFPexpressed from its chromosomal locus under the en-dogenous promoter also revealed an interesting phe-notype, specifically in Skg6-overproducing cells withabnormal cell morphology. For the majority of medium-to large-budded wild-type and Skg6-overproducing cellswith normal morphologies, Hof1–GFP showed a similarlocalization pattern, found either as a single band or as adoublet at the bud neck. However, in 67% of theelongated/multibudded cells overproducing Skg6, Hof1-GFP was absent from the mother-bud neck (Figure 3C).

skg6 suppresses defects of the hof1D mutant: Hof1restricts the septal chitin to the bud neck and promotesseptum formation, which is necessary for cell separationafter actomyosin ring contraction (Vallen et al. 2000).hof1 cells fail to form septa and hence accumulate aschains of cells with a continuous cytoplasm (Lippincott

and Li 1998; Vallen et al. 2000). Since we observed thatSkg6 overproduction impairs Hof1 recruitment to thecell division site, we hypothesized that the cytokinesisdefect of Skg6-overproducing cells may arise fromnegative regulation of Hof1. If such a mechanism exists,deletion of skg6 would be predicted to suppress thephenotype of hof1 mutant cells. To test this hypothesis,we examined genetic interactions among SKG6, TOS2,and HOF1 by tetrad analysis. In the ssd1-d genetic back-ground of our laboratory strains, hof1 cells formed tinyspore colonies and exhibited growth defects at temper-atures ranging from 26� to 37� (Figure 3, D and E). The


tos2 mutation had no effect on this hof1 phenotype(Figure 3, D and E). However, hof1 skg6 cells and hof1skg6 tos2 cells consistently formed spore colonies thatwere of regular size, indicative of normal cellular growth(Figure 3D). These cells also exhibited normal growthat 26� (Figure 3E) and improved growth (relative to hof1cells) at 33� on YEPD agar (not shown). This genetic in-teraction is consistent with observed cytokinesis defectsupon Skg6 overproduction and supports the hypothesisthat Skg6 performs an inhibitory role in cytokinesis,which is antagonistic to the positive role of Hof1.

Skg6 and Tos2 overproduction causes defect in pri-mary septum formation: The results above prompted usto next examine primary septum formation in Skg6-and

Tos2-overproducing cells. Formation of the septumrequires cell-wall synthesis across the bud neck (Bi

2001). To assess deposition of cell wall at the neck, wetreated wild-type and Skg6- or Tos2-overproducingcells with calcofluor white, which stains cell-wall chitin.All wild-type budded cells showed an intensely stainedband at the bud neck, indicative of proper septumformation and closure of the neck (Figure 3F, openarrowheads). In contrast, elongated/multibudded cellsresulting from Skg6 or Tos2 overproduction displayedcalcofluor staining that was extremely faint or absent atthe neck (Figure 3F, solid arrowheads). This phenotypesuggests that the bud necks remained open in these cellsas a result of defective septum formation.

Figure 3.—Effects of Skg6 over-production on Myo1, F-actin, andHof1 localization and primaryseptum formation at the bud neck.(A) Localization of Myo1–GFPin wild-type cells (CCY1710-3C-1)and in cells overproducing HA-Skg6 (CCY1708-1A-1) for 10 hrat 37�. Note e and f, showing theMyo1-GFP signal facing moretoward the mother cell or thedaughter cell, respectively. (B)Organization of the actin cytoskele-ton at the bud neck of wild-typecells (CCY1710-3C) and cells over-producing HA-Skg6 (CCY1708-1A)for 6 hr at 37�. Cells in the bottomrow are included to indicate nor-mal apical polarization of the actincytoskeleton in growing buds. (C)Distribution showing the Hof1-GFPsignal at the bud neck of wild-typecells (CCY1710-3C-2) and cells over-producing HA-Skg6 (CCY1708-1A-2) for 10 hr at 37�. Cells withnormal-sized buds and elongated/multiple buds in the HA-Skg6-overproducing sample werecounted separately. Between 200and 300 cells were counted for eachbud type. (D and E) Deletion ofSKG6 suppresses the growth defectof hof1 cells. A hof1-D101TspHIS5strain (CCY1137-3B) carryingHOF1 on a CEN plasmid wascrossed with a skg6-D1TKan tos2-D2TURA3 strain (CCY1176-11A).Tetrad analysis was performedafter allowing plasmid loss fromthe resulting diploid strain. Sporecolonies bearing the mutationshof1-D101TspHIS5 (CCY1244-4D),hof1-D101TspHIS5 skg6-D1Tkan(CCY1244-7C), hof1-D101TspHIS5

tos2-D2TURA3 (CCY1244-10D), and hof1-D101TspHIS5 skg6-D1Tkan tos2-D2TURA3 (CCY1244-17C) were grown on YEPD agarat 26� for 3 days (D) and then streaked and grown on YEPD agar for 3 days at 26� (E). (F) Visualization of primary septumin wild-type (CCY1710-3C) and HA–Skg6-overproducing (CCY1708-1A) cells by staining the cell wall with calcofluor white. Openarrowheads mark closed septa; solid arrowheads mark open bud necks. HA-Tos2-overproducing cells (CCY1709-2B; not shown)exhibited a septum formation defect similar to HA-Skg6-overproducing cells. All cells are shown at the same magnification.


Taken together, these data suggest that SKG6 andTOS2 when overproduced negatively regulate cytokine-sis by disrupting septum formation. One possible mech-anism for this disruption is their mislocalization of Hof1and/or septins.

Vhs2 and Mlf3 are essential for polarized growth atelevated temperature: We next investigated the geneticroles of the second pair of gic1 gic2 suppressors, VHS2 andMLF3. Vhs2 and Mlf3 proteins are 436 and 452 aminoacids in length, respectively, and their primary sequen-ces share 30% identity and 42% similarity (supplemen-tal Figure 2 at http://www.genetics.org/supplemental/).VHS2 was previously identified as a high-copy suppres-sor of the lethality of hal3 sit4 cells, which are defectivein G1-to-S-phase transition (Munoz et al. 2003). MLF3was identified as a high-copy suppressor of the growthdefects caused by the immunosuppressive drug lefluno-mide (Fujimura 1998). However, the functions of Vhs2and Mlf3 have not been characterized and theirsequences provide no obvious clues regarding theircellular functions. Our identification of VHS2 and MLF3as high-copy suppressors of the gic1 gic2 phenotype sug-gests that they likely contribute to polarized cell growth.

To gain further insights into VHS2 and MLF3 func-tions, we generated deletion strains lacking one or bothgenes. Analysis of the growth phenotypes of vhs2 andmlf3 mutants revealed that neither VHS2 nor MLF3 isessential for the viability of haploid cells grown at arange of temperatures from 13� to 37�, although mlf3cells formed slightly smaller colonies on YEPD agar at37� (not shown). However, vhs2 mlf3 cells were severelyimpaired for growth at 37� (Figure 4A). Interestingly,these defects were more severe in diploids, as homozy-gous diploid vhs2 and mlf3 single-mutant and vhs2 mlf3double-mutant cells exhibited stronger Ts� growth de-fects (and calcofluor white sensitivities; see below) thantheir haploid counterparts (Figure 4A). The enhancedphenotype in diploids compared to haploids has beenreported for other cell polarity mutants, including bni1and msb3 msb4 (Bi et al. 2000).

When incubated at 37� in liquid YEPD medium,diploid vhs2 mlf3 cells became increasingly large andround over time, a phenotype suggesting loss of polarity

(Figure 4B). However, unlike cdc42 or gic1 gic2 cells(Chen et al. 1997; Johnson 1999), vhs2 mlf3 cells did notarrest exclusively as unbudded cells. Instead, an enrich-ment of unbudded vhs2 mlf3 cells occurred graduallyafter shift to the nonpermissive temperature (Figure5B). Unbudded vhs2 mlf3 cells that appeared large andround also showed a characteristic ‘‘wrinkled’’ morphol-ogy (Figure 4B, arrowhead), suggesting that loss of cellpolarity may lead to weakening of the cell wall and lysisof these cells. As expected, the morphological defects(loss of polarity and ‘‘wrinkled’’ appearance) were muchweaker in diploid vhs2 and mlf3 single-mutant cellscompared to diploid vhs2 mlf3 double-mutant cells(Figure 4B).

We next localized Vhs2-GFP and Mlf3-GFP fusionproteins expressed from low-copy plasmids under con-trol of the ACT1 promoter. These constructs comple-mented the temperature-sensitive growth defects ofvhs2 mlf3 double-mutant cells (not shown), indicatingthat they are functional. In both cases, cytoplasmiclocalization was observed (Figure 4C). Similar resultswere obtained for chromosomally tagged GFP fusions ofVHS2 and MLF3 expressed from their endogenouspromoters (not shown).

Actin organization defects in vhs2 mlf3 cells: Sincea polarized actin cytoskeleton (patches and cables)is required for polarized cell growth (Pruyne andBretscher 2000b), the morphological defect of vhs2mlf3 cells prompted us to examine actin organizationin these cells. When grown at 37�, �50% of unbuddedwild-type diploid cells had a polarized pattern of actinpatches that were concentrated at one end of the cell(Figure 5, A and C), indicative of cells poised for budemergence. Essentially all small-budded wild-type cellshad a highly polarized pattern of actin organization,with actin patches being concentrated in the small buds(Figure 5, A, open arrowheads, and D). In contrast,diploid vhs2 mlf3 cells showed depolarized actin orga-nization, which intensified with increasing time of incu-bation at 37�. After 7.5 hr growth at 37�, only �2% ofunbudded cells showed a polarized distribution of actinstructures (Figure 5C). Furthermore, �25% of small-budded vhs2 mlf3 cells had actin patches concentrated

TABLE 3

Distribution of the actin cytoskeleton at the mother-bud neck of cells with elongated buds

Actin localization at the mother-bud neck of cells with elongated buds

Protein overproduced None Normal Diffused

HA-Skg6 28 10 62HA-Tos2 73 7 20

Haploid wild-type (CCY1710-3C) cells and cells overproducing Skg6 (CCY1708-1A) or Tos2 (CCY1709-2B)were cultured at 26� in YEP-raffinose (2%) medium to log phase. Galactose (4%) was then added to the growthmedium to induce overproduction of HA-Skg6 and HA-Tos2 and cultures were incubated further at 37� for 6 hr.Cells were fixed with formaldehyde (3.7%), stained with rhodamine–phalloidin, and examined by fluorescentmicroscopy. None of the wild-type cells formed elongated buds and therefore are not listed.


in the mother, with a complete loss of patches from thebud (Figure 5, A, solid arrowheads and inset, and D).This subset of cells had atypically round and largemother-cell bodies, suggesting that abnormal F-actindistribution may have led to inappropriate expansion ofthe mother-cell bodies at the expense of bud growth. Inaddition, the majority of budded vhs2 mlf3 cells thatshowed a fairly normal polarized distribution of actinhad an unusually large number of actin patches in themother-cell bodies, indicating that the actin cytoskele-ton in these cells might not be fully polarized. Together,these data suggest that the combined loss of Vhs2 andMlf3 impairs polarized actin organization, which in turndisrupts polarized growth.

Defect in mating projection formation in vhs2 mlf3cells: Next, we examined whether the actin polarizationdefect of vhs2 mlf3 cells was reflected in their ability toform mating projections upon pheromone treatment.

At 26�, MATa haploid vhs2 mlf3 cells responded to a-factor in a manner similar to wild-type cells and initiatedthe formation of mating projections. At 37�, however,unlike wild-type cells that formed elongated matingprojections, vhs2 mlf3 cells became somewhat larger androunder with a pointed structure restricted to one sideof the cell surface that apparently failed to elongate as amating projection (Figure 6). This result further re-inforces the roles of Vhs2 and Mlf3 in cell polarization.

Cell-wall defects in vhs2 mlf3 cells: We noted thatafter 5 hr of growth at 37�, vhs2 mlf3 cell cultures becamevisibly flocculent and contained small clumps of cellsthat were not separable by mild sonication (Figure 4B).This observation, taken together with the wrinkled cellmorphology mentioned above, suggests that vhs2 mlf3cells may have cell-wall defects. To investigate this pos-sibility further, we used two complementary assays. First,we compared the sensitivity of vhs2 and mlf3 single-mutant

Figure 4.—(A) Growth of congenic haploid and homozygous diploid wild-type (CCY1292-1A and DBY1830), vhs2 (CCY1292-4Band CCY1720), mlf3 (CCY1292-1D and CCY1721), and vhs2 mlf3 (CCY1292-5B and CCY1338) cells on YEPD agar, YEPD agar con-taining 1 m sorbitol, and YEPD agar containing calcofluor white (CFW). Suspensions of cells were spotted on the respective agarplates and incubated at indicated temperatures for 2 days. (B) Morphology of congenic diploid wild-type (DBY1830) and homo-zygous vhs2 (CCY1720), mlf3 (CCY1721), and vhs2 mlf3 (CCY1338) cells. Cells were grown to logarithmic phase in YEPD mediumat 26�, shifted to 37� for the indicated time periods, fixed with 3.7% formaldehyde, and imaged using phase-contrast microscopy.The arrowhead in the vhs2 mlf3 panel highlights a cell with the wrinkled morphology. (C) Cytoplasmic localization of Vhs2 andMlf3. Diploid homozygous vhs2 mlf3 (CCY1338) cells carrying empty vector (pTD125), GFP-VHS2 (pCC1658), and GFP-MLF3(pCC1659) were cultured in minimal medium lacking uracil (for maintaining plasmid) to logarithmic phase and washed oncewith PBS before visualization. All cells are shown at the same magnification.


and vhs2 mlf3 double-mutant cells to calcofluor white,which interferes with cell-wall assembly and exacerbatesthe growth defects of strains defective in this process(Ram et al. 1994). Second, we tested whether addition ofsorbitol (an osmotic stabilizer) to the growth mediumcould alleviate growth defects of the mutants. Sorbitolis known to suppress the cell lysis defects of some cell-wall integrity mutants, including those defective in theRho1-Pkc1 pathway (Kamada et al. 1996; Heinisch et al.1999). Our results showed that, in a haploid back-ground, vhs2 mlf3 double-mutant cells were super-sensitive to calcofluor white (Figure 4A). In a diploidbackground, this effect was exacerbated so that evenvhs2 and mlf3 single-mutant cells were supersensitive tocalcofluor (Figure 4A). In addition, the growth defectsof mlf3 and vhs2 mlf3 mutants at 37� were almostcompletely rescued by addition of 1 m sorbitol to thegrowth medium (Figure 4A). Since the onset of theactin depolarization phenotype precedes the wrinkledmorphology phenotype (Figure 4B and Figure 5, C and

D), we believe that the cell-wall defect in vhs2 mlf3 cellslikely occurs as a consequence of actin depolarization.Consistent with this, we found that providing osmoticsupport in the growth medium is not sufficient toprevent the actin polarization defects of vhs2 mlf3 cells.The number of unbudded cells with depolarized actincytoskeleton and small-budded cells with buds devoidof actin patches remained roughly similar in vhs2 mlf3cultures grown in the presence or absence of 1 m

sorbitol (not shown).All of these data are consistent with vhs2 mlf3 mutants

being defective in actin polarization at 37�, leading todefects in cell-wall assembly and cell lysis, which in turncauses the wrinkled cell morphology.

GIC1 and G1 cyclin genes as high-copy suppressorsof vhs2 mlf3 cells: We next investigated the functionalrelationships of Vhs2 and Mlf3 with other proteinsknown to regulate polarized cell growth. Toward thisend, we individually overexpressed 59 different ‘‘polar-ity’’ genes to test their effects on the Ts� growth of

Figure 5.—Actin polarization defect in vhs2 mlf3 cells. Diploid wild-type (DBY1830) and homozygous vhs2 mlf3 (CCY1338) cellsincubated at 37� for the indicated time periods were fixed in formaldehyde and stained with rhodamine–phalloidin to examineF-actin organization. (A) Phase-contrast and actin-staining images of the same cells after a 5-hr incubation at 37�. Open arrow-heads show small-budded wild-type cells with actin patches concentrated in the bud, while solid arrowheads point to small-buddedvhs2 mlf3 cells devoid of actin patches in the bud. All cells are shown at the same magnification except in the inset. Phenotypeswere scored in cell populations and graphed as total unbudded cells (B), unbudded cells with polarized actin patches (C), andsmall-budded cells lacking actin patches in the bud (D). Two hundred or more cells were counted for each sample.


haploid vhs2 mlf3 cells (for a complete list of genestested, see supplemental Figure 3 at http://www.genetics.org/supplemental/). We identified GIC1 (and to alesser extent GIC2) as an efficient high-copy suppressorof the vhs2 mlf3 growth defect at 37� (Figure 7A). SinceVHS2 and MLF3 also are high-copy suppressors of gic1gic2 cells (Figure 1A), this pattern of reciprocal sup-pression suggests that Vhs2 and Mlf3 may function in apathway that is genetically redundant with a Gic1/Gic2pathway. Consistent with this interpretation, vhs2 mlf3gic1 gic2 cells showed a more severe Ts� growth defectthan vhs2 mlf3 or gic1 gic2 cells (Figure 7B).

We also identified the G1 cyclins CLN1, CLN2, andPCL1 as robust high-copy suppressors of vhs2 mlf3 cells(Figure 7A). Cln1 and Cln2 associate with Cdc28 to forman active Cdk–cyclin complex that promotes G1-to-S-phase transition. Likewise, Pcl1 and Pcl2 associate withPho85 to form an active Cdk–cyclin complex thatpromotes G1-to-S phase transition, and this complexbecomes essential in the absence of Cln1 and Cln2(Measday et al. 1997). The identification of G1 cyclingenes as high-copy suppressors of vhs2 mlf3 cellsprompted us to investigate whether vhs2 mlf3 mutantsare defective in G1-to-S transition. FACS analysis of vhs2mlf3 cells released from a-factor (G1) arrest into the cellcycle at 37� revealed that they entered S phase normallyand progressed through the cell cycle with wild-typekinetics (not shown). This result suggests that the abilityof G1 cyclins to suppress vhs2 mlf3 growth defects maynot be related to their role in promoting cell cycleprogression. Instead, suppression may stem from theadditional roles of these cyclins in regulating Cdc42-

related polarity functions. Both Cdc28-Cln1/2 andPho85-Pcl1,2 are required for phosphorylation ofCdc24, the guanine nucleotide exchange factor forCdc42, and for assembly of the septin ring (Moffat andAndrews 2004). In addition, several lines of geneticevidence suggest that Pho85–Pcl1,2 complexes promoteCdc42 activity or a process that substitutes for Cdc42function (Lenburg and O’Shea 2001). Since Cdc42 is amaster regulator of polarized actin assembly and cellgrowth, increased levels of these cyclins may act throughCdc42 to restore growth to vhs2 mlf3 cells at 37�.

DISCUSSION

Here, we identified two pairs of structurally relatedgenes, SKG6 and TOS2 and VHS2 and MLF3, asmulticopy suppressors of the growth and actin organi-zation defects of gic1 gic2 cells. One of our initial goals inperforming a dosage suppression screen was to gaininsights into how Gic1 and Gic2 regulate the actincytoskeleton and polarized growth. Although our anal-yses have not yet clarified this mechanism, they

Figure 6.—vhs2 mlf3 cells exhibit defects in formation ofelongated mating projections. Wild-type (CCY1292-1A) andvhs2 mlf3 (CCY1292-5B) haploid cells were cultured in acidicYEPD medium (containing 0.1 m citrate, pH 4.5) at 26� andarrested as unbudded cells with a-factor (10 mg/ml). When.90% of the cells were arrested (shown in the 26� panel),a second dose of a-factor was added to continue the cell cyclearrest and cultures were shifted to 37� for 4 hr (shown in the37� panel). Cells were fixed and observed by phase-contrastmicroscopy.

Figure 7.—(A) High-copy-number plasmids expressingGIC1 and G1 cyclins suppress the growth defect of vhs2 mlf3cells. Suspensions of haploid vhs2-D1TTRP1 mlf3-D1Tkancells (CCY1292-10C) carrying different 2m plasmids were spot-ted onto YEPD agar and allowed to grow at the indicated tem-peratures for 2 days. The plasmids used were pRS426 (emptyvector control), YEp24-CLN1 (CLN1, gift from F. Cross),YEp24-CLN2 (CLN2, gift from F. Cross), pBA531 (PCL1, giftfrom B. Andrews), pCC904 (GIC1), pCC1606 (VHS2),pCC1635 (MLF3), pCC967 (GIC2), and pBA623 (PCL2, giftfrom B. Andrews). (B) The mutant phenotype of gic1 gic2 cellsis exacerbated by additional mutations in VHS2 and MLF3.Haploid wild-type (DBY1829), gic1 gic2 (CCY1726-18C), gic1gic2 vhs2 (CCY1726-2B), gic1 gic2 mlf3 (CCY1726-5B), andgic1 gic2 vhs2 mlf3 (CCY1726-4D) cells were spotted ontoYEPD agar and allowed to grow at the indicated temperaturesfor 2 days.


demonstrate the involvement of both new gene pairs incell functions that involve Gic1 and Gic2: cytokinesis(SKG6 and TOS2) and actin organization and polarizedcell growth (VHS2 and MLF3). Below, we discuss eachgene pair separately.

Skg6 and Tos2 help regulate cytokinesis and theswitch from apical-to-isotropic cell growth: All of ourdata characterizing Skg6 and Tos2 lead us to proposethat they have an inhibitory function in cytokinesis,primarily via regulation of septin organization and/orHof1 localization and, in turn, septum formation. Thisis supported by the relocalization of Skg6-GFP andTos2-GFP from the bud tip to the bud neck precedingcytokinesis (Drees et al. 2001 and our unpublisheddata) and by the hyperelongated- and multiple-budphenotype of cells overexpressing Skg6 or Tos2 (Figure2A), their failure to undergo cell division (Figure 2C),and their mislocalization and/or disrupted organiza-tion of septins, Myo1, actin, and Hof1 at the bud neck(Figure 2D; Figure 3, A–C). We favor the model thatSkg6 and Tos2 act through Hof1 and septins to affectcytokinesis rather than Myo1, because we observed noobvious defects in recruitment or closure of the acto-myosin ring. Further, deletion of SKG6 suppressed thetemperature-sensitive growth of hof1 cells (Figure 3, Dand E) and septum formation was impaired in multi-budded Skg6- or Tos2-overproducing cells (Figure 3F).

Previous studies have suggested that Gic1 and Gic2may regulate cytokinesis on the basis of localization ofGic proteins at the bud neck in large-budded cells andthe synthetic lethal interaction between gic1 gic2 andcla4 (Chen et al. 1997). Further, a recent study showedthat Gic1 and Gic2 are required for the ability of Cdc42to recruit and organize the septin collar at the bud neck(Iwase et al. 2005). The similar localization pattern ofSkg6 and Tos2 to that of Gic1 and Gic2 and their co-implication in regulating cytokinesis may explain theiridentification as suppressors of gic1 gic2 mutations.

Although we have primarily focused on the functionsof Skg6 and Tos2 in cytokinesis, it may not be theirexclusive function, given their localization to sites ofbud emergence and as a polarized cap in medium- tolarge-budded cells. Skg6 and Tos2 may have additionalroles in polarized growth—possibly in regulating theswitch from apical to isotropic cell growth, as inferredfrom the elongated bud phenotype caused by their over-production (Figure 2A and Table 2). In fact, Toenjes

et al. (2004) reported an elongated cell phenotype uponTOS2 overexpression and suggested that this phenotypemay result from prolonged retention of Cdc24 and/orother polarity factors at sites of polarized growth.

Vhs2 and Mlf3 help regulate actin cytoskeletonorganization, cell-wall integrity, and polarized cellgrowth: Normal polarized morphogenesis in S. cerevisiaerequires orientation of the actin cytoskeleton, which inturn targets secretory vesicles to deliver membrane andcell-wall synthesis/modifying enzymes toward regions of

active growth. The importance of regulating this processis underscored by the fact that�27% of SBF target genes(those expressed during late G1 when cell polarity isestablished) are involved in cell-wall biogenesis/main-tenance and/or polarized growth (Iyer et al. 2001). Ourresults have implicated Vhs2 and Mlf3 in this process.vhs2 mlf3 cells have a depolarized actin cytoskeleton andmorphological phenotypes, indicating loss of polarity(Figures 5 and 4B, respectively). For example, vhs2 mlf3cells become enriched in depolarized unbudded cells.This phenotype is not likely to result from cell arrest inG1 since we did not find any obvious defects in cell cycleprogression. Instead, we suspect that vhs2 mlf3 cells maybe impaired in apical polarization of the actin cytoskel-eton. This is suggested further by our observation thatsmall-budded vhs2 mlf3 cells are completely devoid ofvisible F-actin structures in the bud (Figure 5A, insert,and D). In addition, vhs2 mlf3 cells are defective informing normal elongated mating projections (Figure6). Thus, Vhs2 and Mlf3 clearly appear to have roles inregulating actin cytoskeleton organization and cellpolarity. However, both of these proteins are unlikelyto be stably associated with components of the actincytoskeleton on the basis of their diffused cytoplasmiclocalization (Figure 4C).

Our data also demonstrate Vhs2 and Mlf3 involve-ment in regulating cell-wall integrity. However, threeobservations suggest that this function is likely to beindirect, stemming from the roles of Vhs2 and Mlf3 inregulating actin polarization. First, in vhs2 mlf3 cells theonset of actin polarization defects precedes the onset ofcell lysis defects. Second, addition of sorbitol to thegrowth medium rescues the cell-wall defects but not theactin polarization defects of vhs2 mlf3 cells. Third, vhs2mlf3 cells accumulate as large and round cells, whereasall known mutants of the cell integrity pathway lyse anddie at the small-budded stage (Heinisch et al. 1999).

Vhs2 and Mlf3 functions in regulating actin organi-zation are redundant with Gic1 and Gic2: AlthoughVHS2 and MLF3 overexpression suppresses the Ts�

growth and actin polarization defects of gic1 gic2 cells,multiple lines of evidence presented here suggest thatVhs2 and Mlf3 function in a pathway that is distinct andparallel to Gic1 and Gic2:

1. We have demonstrated reciprocal high-copy suppres-sion between these gene pairs; gic1 gic2 is suppressedby VHS2 and MLF3, and vhs2 mlf3 is suppressed byGIC1 (Figures 1 and 7A). This is not expected forgenes that function in the same linear pathway.

2. The growth phenotype of gic1 gic2 cells is exacer-bated by deletion of VHS2 and MLF3 (Figure 7B).

3. Gic1 and Gic2 localize to sites of polarized growth(Chen et al. 1997), whereas Vhs2 and Mlf3 arecytosolic (Huh et al. 2003 and Figure 4C).

4. The Ts� growth defects of vhs2 mlf3 but not gic1 gic2cells are rescued by sorbitol.


5. Haploid gic1 gic2 cells but not vhs2 mlf3 cells exhibitdefects in bud-site selection.

Thus, all lines of available evidence point to Vhs2 andMlf3 having functions that are separate from Gic1 andGic2, operating in a parallel (and perhaps partiallyoverlapping) pathway directing polarized growth. Oneof the only common phenotypes shared by gic1 gic2and vhs2 mlf3 mutant cells is defective actin cyto-skeleton polarization, and perhaps this point of similar-ity provides the basis for their reciprocal high-copysuppression.

To summarize, we have characterized the genetic andcellular roles of two pairs of proteins whose functionsuntil now were unknown. We show that Skg6 and Tos2regulate organization of proteins (septins, actin, andHof1) at the bud neck to influence septum formationand cytokinesis, while Vhs2 and Mlf3 regulate polariza-tion of the actin cytoskeleton and cell growth. A moreprecise understanding of the cellular functions of eachof these proteins awaits identification of their in vivoligands and determination of their biochemical activi-ties. However, we have demonstrated the involvement ofthese proteins in specific physiological processes anddefined their genetic interactions, providing a strongfoundation for future analyses.

We are grateful to Guang-Chao Chen and Liaoteng Wang for theinitial isolation of multicopy suppressors. We thank James Moseley andAvital Rodal for critical reading and editing of the manuscript. Thiswork was supported by a National Institutes of Health (NIH) grant(GM63691) to B.L.G. and an NIH grant (GM45185) and a TexasHigher Education Coordinating Board advanced research programgrant (003658-0427b) to C.S.M.C.

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Communicating editor: M. D. Rose


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