INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Polarized cell division is a fundamental process in which cells divide along specific cleavage planes; this process is essential for the development of both eukaryotes and prokaryotes. Polarized cell divisions can mediate appropriate cell-cell contacts and partition cytoplasmic components asymmetrically between daughter cells. Polarized cell divisions occur during the life cycle of many organisms, including early embryogenesis in Caenorhabditis elegans (Hyman and White, 1987), neurogenesis in Drosophila and mammals (Kraut et al., 1996), spore development in Bacillus subtilus (Shapiro, 1993), and development of the snail body plan (Freeman and Lundelius, 1982). The mechanisms for selecting sites for polarized growth and division as well as for directing growth toward these sites are only beginning to be understood.

The budding yeast Saccharomyces cerevisiae is an excellent model for studying polarized cell division. S. cerevisiae exists in different forms, each with a specific cell morphology and division pattern (Roemer et al., 1996b; Costigan and Snyder, 1998; Madden and Snyder, 1998). During growth in rich media, both haploid and diploid yeast cells are ellipsoid and select bud sites according to their mating locus (MAT) and pedigree (Freifelder, 1960; Snyder, 1989; Chant and Pringle, 1995). Haploid MATa and MAT cells use an axial budding pattern in which cells bud adjacent to the preceding site of cytokinesis (i.e., the proximal poles). Diploid MATa/MAT cells exhibit a more complex bipolar budding pattern: daughter cells bud at distal poles (180° from the birth site), whereas mother cells bud either distal or proximal to the birth site, with new mothers exhibiting a bias for budding at the distal site (i.e., near the previous site of cytokinesis). The different budding patterns are thought to fulfill different purposes. The diploid bipolar pattern is thought to maximize exposure of a growing microcolony to nutrients (Gimeno and Fink, 1992; Gimeno et al., 1992; Madden et al., 1992); in contrast, the haploid pattern is thought to position cells in a microcolony derived from a homothallic cell that switches its mating type close to one another to facilitate mating and diploid formation (Nasmyth, 1982). It is thought that cortical tags mark future bud sites and serve as spatial cues for the initiation of new buds (Chant et al., 1991; Snyder et al., 1991; Flescher et al., 1993; Chant and Pringle, 1995).

Three distinct classes of proteins important for bud site selection have been identified (Snyder, 1989; Chant and Herskowitz, 1991). The first class is specifically required for the axial pattern. Mutations in genes encoding any of these proteins result in bipolar budding in haploid cells, but they do not disrupt the diploid budding pattern. It is thought that these genes are important for tagging the axial pole and/or recognizing the axial tag (Chant and Herskowitz, 1991; Flescher et al., 1993; Fugita et al., 1994; Chant et al., 1995; Halme et al., 1996; Roemer et al., 1996a; Sanders and Herskowitz, 1996). The second class is specifically required for the bipolar pattern. It is likely that these proteins are involved in marking the poles of diploid cells at sites for budding or helping direct those tags to their appropriate location (Snyder, 1989; Bauer et al., 1993; Zahner et al., 1996; Chen et al., 2000; Sheu et al., 2000). The third class is required for both the axial and bipolar patterns. These gene products are believed to recognize the axial or bipolar tags provided by the first two classes of gene products and convey this information to the machinery involved in establishing cell growth (Bender and Pringle, 1989; Chant and Herskowitz, 1991; Chant et al., 1991; Park et al., 1993). Although much is known about the molecular mechanisms that help mediate the axial pattern of budding, much less is known concerning the mechanisms of the bipolar pattern.

A number of components important for bipolar bud site selection have been identified and characterized (Herskowitz et al., 1995; Pringle et al., 1995; Drubin and Nelson, 1996; Costigan and Snyder, 1998;). Three genes, BUD8, BUD9, and STE20, when mutated, cause diploid cells to form buds at one pole (Zahner et al., 1996; Sheu et al., 2000). bud8/bud8 and ste20/ste20 cells bud at the proximal pole of the daughter cell, whereas bud9/bud9 cells bud at the distal pole. Bud8 and Bud9 have been proposed to act as bipolar landmarks or tags that recruit components involved in bud formation (Zahner et al., 1996). Consistent with this hypothesis, Bud8 localizes at the distal pole; two different localizations of Bud9 have been reported, one at the distal pole (where it would most likely be a repressor of bud formation) and the other at the proximal poles (where it might serve as a tag) (Taheri et al., 2000; Harkins et al., 2001). Ste20 is a PAK protein kinase homologue that has been shown to lie in the same genetic pathway as Bud8 (Sheu et al., 2000). How tags are produced, processed, and localized to the poles has not been investigated.

The actin cytoskeleton is essential for both polarized growth and bipolar bud site selection and is thought to mediate directional transport of secretory vesicles (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Novick and Botstein, 1985; Mulholland et al., 1994; Drubin and Nelson, 1996; Pruyne et al., 1998). Both cortical actin patches and components of the secretory apparatus localize to sites of growth (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Finger and Novick, 1998). Mutations in genes encoding actin, several actin-associated proteins, or proteins involved in actin organization cause defects in bipolar budding similar to those of act1 cells (Drubin et al., 1990; Adams et al., 1991; Crouzet et al., 1991; Vojtek et al., 1991; Bauer et al., 1993; Holtzman et al., 1993; Sivadon et al., 1995; Zahner et al., 1996).

A variety of other nonessential polarity proteins are also required for bipolar bud site selection. Deletion of SPA2, PEA2, BUD6, or BNI1 cause a random budding defect. Spa2, Pea2, Bud6, and Bni1 form a complex, the 12S polarisome, that localizes to sites of polarized growth (Fujiwara et al., 1998; Sheu et al., 1998); these proteins are present as a patch at the incipient bud site, at the tip of the growing bud, and at the mother-bud neck region before cytokinesis (Snyder, 1989; Gehrung and Snyder, 1990; Snyder et al., 1991; Kohno et al., 1996; Valtz and Herskowitz, 1996; Zahner et al., 1996; Amberg et al., 1997; Evangelista et al., 1997). Spa2p, Pea2p, Bud6p, and Bni1p are each required for apical growth (Sheu et al., 2000), which is the initial phase of bud growth in which cells grow at the bud tip (Lew and Reed, 1993). It has been suggested that these proteins are required for the polarized deposition of the distal tag during this period (Sheu et al., 2000).

Understanding bipolar bud site selection and identifying the molecules that participate in this process will provide insight into the molecular mechanisms of how this process is carried out and coordinated with other cellular events. Recently, a set of diploid strains containing homozygous deletions of nonessential yeast open reading frames (ORFs) was generated; this set allows a genome-wide investigation into the bipolar bud site selection (Winzeler et al., 1999). Here we report a genome-wide systematic screening of 4168 homozygous diploid mutants (affecting 84% of the total nonessential annotated ORFs) for those defective in the bipolar budding pattern. These studies led to the identification of 127 genes involved in this process, of which only 16 have been reported to affect bipolar budding previously. Based on the classes of mutants identified and the subsequent analysis of these mutants, we have identified a variety of cellular processes involved in the bipolar pattern and present a global model of how this process occurs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Yeast Strains and Media

All strains used in this study are congenic derivatives of FY2, which is itself a direct descendant of S288C (Brachmann et al., 1998). Most of the strains contain precise deletions in which the entire ORF after the ATG is replaced with a kanMX4 marker. Two independent transformants were prepared and MATa and MAT segregants were recovered from each transformant and mated to form the homozygous diploid strains (Winzeler et al., 1999). The different strains are available from Research Genetics (Huntsville, AL). Growth media and genetic manipulation have been described previously (Sherman et al., 1986; Guthrie and Fink, 1991).

Budding Pattern Analysis

Mutant strains were grown individually in 0.8 ml of liquid YPAD in 96-well format boxes (Dot Scientific, Burton, MI) and incubated at 30°C with shaking. Each well of the box contained one 3.5-mm glass bead to facilitate mixing. Cells were first grown to stationary phase and then diluted into another 96-well box with glass beads; cells were grown for at least six generations until mid-log-phase. Mid-log-phase cells were then fixed by the addition of formaldehyde to a final concentration of 3.7%. Cells were subsequently incubated for 1 h at 30°C, washed with phosphate-buffered saline, and resuspended in phosphate-buffered saline. Calcofluor White (Sigma, St. Louis, MO) was added to a final concentration of 2 µg/ml, bud scars and birth scars were visualized by fluorescence microscopy. Birth scars are chitin-depleted structures that mark the region where mother cells separated from their daughter cells. A bud site is defined as proximal, distal, or medial depending on whether the bud or bud scar resides in the one-third portion of the cell closest to the birth scar, the one-third of the cell opposite the birth scar, or the middle-third of the cell, respectively (Flescher et al., 1993). Bud site selection mutants were identified and retested. All of the unipolar and axial-like mutants were tested for mating type and appropriate auxotropic markers and examined for cell size to ascertain whether they are diploid or haploid. Twenty-eight strains were found to be haploids. Only mutants that reproducibly exhibited a budding pattern defect and were not haploids were chosen for further analysis.

To quantify the fidelity of bipolar bud site selection, only cells that had experienced at least three budding events were examined. Cells with bud scars exclusively at one pole were classified as unipolar; cells with medial bud scars were scored as random budding. The percentage of random budding cells was then determined for each sample. For each sample, 100 cells were counted from at least four independent fields. For some mutant strains, bud site selection was scored for the first, second, and third bud divisions with the use of the criteria described (Flescher et al., 1993; Zahner et al., 1996). For these analyses a total of 200-600 cells from each sample were scored for each division type.

To determine whether the genes required for the bipolar pattern are also important for the haploid axial pattern, 104 MATa deletion strains were examined by Calcofluor staining. For the genes whose loss in diploids caused a unipolar pattern, both MATa and MAT strains were examined.

Examination of Cell Morphology

The cells used for morphological analysis were grown as described above. Cells were examined by phase-contrast and differential interference contrast microscopy, and mutants were classified as described in RESULTS.

Construction of GFP-tagged Yeast Strain

GFP-tagged fusion proteins were constructed in MATa and MAT haploid wild-type yeast strains with the use of the strategy described by Schneider et al. (1995). Primers were designed with the use of the N-terminal coding sequence of the gene of interest and used to amplify a region of the plasmid pMPY-GFP (a generous gift from Jun-Yi Leu). The resulting ~2.0-kb polymerase chain reaction (PCR) product contains the complete URA3 gene flanked by direct repeats encoding the GFP gene; the entire cassette is flanked by 45 bp of the gene sequence. These DNA fragments were transformed into haploid yeast, and transformants that had integrated the fragment correctly at the chromosomal locus were identified by PCR analysis. The integrants were allowed to grow overnight in YPAD and plated onto medium containing 5-fluorolorotic acid; resistant colonies that had lost the URA3 gene through homologous recombination between the two GFP-coding sequences were identified by PCR. This recombination event leaves a single in-frame GFP-coding sequence after the ATG codon. MATa and MAT strains containing the in-frame GFP cassette were then mated, and homozygous tagged diploid strains were analyzed to determined whether the GFP allele was functional. Homozygous ORF-GFP/ORF-GFP diploid strains were examined for growth rate and bipolar budding pattern defects.

GFP-Fluorescence Microscopy

Yeast strains harboring the GFP-Bud8 plasmid or strains with GFP-tagged Bud proteins were grown in synthetic complete medium lacking particular nutrients or synthetic complete medium to mid-log-phase. Cells from 3-ml cultures were harvested and immediately viewed with the use of a fluorescence microscope (Leica, Deerfield, IL) equipped with a GFP filter set (41017, Endow GFP, Chroma Technology, Brattleboro, VT). For similar experiments, all images were processed identically with the use of Photoshop 5.0 (Adobe Systems, Mountain View, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of Genes Important for Bipolar Budding in Yeast

To identify mutants that specifically affect the bipolar budding pattern, we examined 4168 homozygous diploid deletion strains disrupted in different nonessential ORFs by staining with Calcofluor White. Calcofluor binds the chitin-rich bud scars that remain on the cell surface after cytokinesis and thus serve as markers of sites of previous cell divisions (Hayashibe and Katohda, 1973). Yeast cells were grown to mid-log phase, fixed, and stained with Calcofluor, and the budding pattern and cell morphology of each strain were examined. Wild-type diploid yeast cells undergo bipolar budding and bud scars are usually found at both poles of the cell (Figure 1). We identified 127 mutants that reproducibly displayed altered budding patterns; these mutants fell into three categories: unipolar, axial-like, and random (Table 1). In the unipolar mutants, bud scars lay at either the proximal or the distal pole of the cell. In the axial-like mutants, cells displayed chains of adjacent bud scars reminiscent of those observed for haploid cells; the bud scars in these classes usually initiated at the proximal pole but sometimes occurred in the equatorial region or at the distal pole. In the random mutants, bud scars appeared distributed over the entire surface of the cells. Of the 127 mutants identified, 105 are disrupted for genes that have been previously characterized; 22 affected uncharacterized ORFs. Twenty of the new genes were named BUD13-BUD32 (Tables 2-4).

View larger version (60K): [in this window] [in a new window]   Figure 1.   The budding pattern of diploid wild-type (WT) and unipolar mutant strains. Cells of the indicated strains were stained with Calcofluor to visualize bud scars.

Unipolar Mutants

Ten mutants were found that exhibited a unipolar budding pattern (Table 2). Seven of the mutants have a strong phenotype in which >70% of the cells exhibited a unipolar budding pattern when cells with more than three bud scars were scored. Three exhibit a partial phenotype in which 50-70% of the cells exhibit the defect (Table 2).

BUD8, BUD9, and STE20. Of the 10 mutants three, bud8/bud8, bud9/bud9, and ste20/ste20, were known previously to exhibit a unipolar budding pattern (Zahner et al., 1996; Sheu et al., 2000; see INTRODUCTION). bud8/bud8 and ste20/ste20 cells bud at the proximal pole, whereas bud9/bud9 mutants bud at the distal pole.

IST3 and BUD13/YGL174w. IST3 is a gene that has been described to have a role in mediating sodium tolerance; however, its molecular role is not known (Entian et al., 1999). YGL174w is an unknown gene that we named BUD13. These two proteins were recently reported to interact in vivo by the yeast two-hybrid assay (Uetz et al., 2000). Mutants lacking these genes display a unipolar budding pattern, with bud scars clustered adjacent to the birth scar at the proximal pole (Figure 1). In these mutants, individual bud sites can be observed adjacent to, overlapping, or within the birth scar and in the vicinity of other bud scars; however, these bud scars are not always immediately adjacent to the bud site of the previous cell cycle and are more loosely distributed in the polar region. This distribution is typical of the bipolar pattern at the proximal pole and is distinct from the axial budding pattern used by wild-type haploid cells. In addition, ist3/ist3 and ygl174w/ygl174w exhibit elongated cell shapes; this phenotype is distinct from bud8/bud8 and ste20/ste20 mutants, which are round and exhibit an apical growth defect (Sheu et al., 2000).

ALG5, ALG6, ALG8, and ALG10. ALG5, ALG6, ALG8, and ALG10 are a group of genes whose products function early in the yeast dolichol pathway that synthesizes the dolichol-linked oligosaccharide precursor for N-linked protein glycosylation (Heesen et al., 1994; Stagljar et al., 1994; Reiss et al., 1996; Burda and Aebi, 1998). ALG5 encodes a dolichol-P-glucose synthetase, and ALG6, ALG8, and ALG10 are genes encoding glucosyltransferases that, respectively, transfer the first, second, and third -1,3-linked glucose to Dol-PP-GlcNac2Man9 in the final modification of outer chain elongation of N-linked oligosaccharides. Mutations of these genes block the addition of glucose residues to the lipid-linked oligosaccharide, but the nonglucosylated oligosaccharides are still transferred to proteins (Huffaker and Robbins, 1983). Homozygous deletion strains lacking ALG5, ALG6, ALG8, or ALG10 have a round cell shape and a partial unipolar distal budding pattern (74, 85, 56, and 53%, respectively) (Table 2; Figure 1). These results indicate that N-linked glycosylation is required for proper bud site selection.

LSM6. Lsm6 is a member of the Sm-like group of proteins and has been implicated in mRNA decay and the function of the U6 snRNP (small nuclear ribonucleoprotein particles), which is involved in mRNA splicing (Tharun et al., 2000). Deletion of LSM6 causes most cells to undergo unipolar distal budding (69.5% distal budding; 23.4% bipolar budding; 7.1% random budding; Figure 1). Interestingly, deletion of LSM1, which only affects mRNA decay and not U6 function (Tharun et al., 2000), causes a random budding phenotype (see below). Presumably, Lsm6 regulates the stability or splicing of an RNA transcript important for mediating the bipolar budding pattern, such as Bud8.

Axial-like Mutants

Five mutants were identified that exhibit an axial-like budding pattern (Table 3). This pattern differs from the unipolar pattern in that the cells had long chains of bud scars, very similar to those seen on axial budding cells (Chant and Pringle, 1995), indicating that new buds formed next to the previous site, rather than simply clustering at one end of the cell. However, in contrast to normal axially budding cells, these mutants produced chains of bud sites starting at either the distal pole or the proximal pole, as well as in the equatorial region, although there was a strong bias in most cases for one end of the chain to originate from the proximal pole. The bud scar chains can be very long and sometimes extend from one pole to another.

BUD7. BUD7 was previously identified in a visual screen for mutants with a defect in the bipolar budding pattern; it was described as having a heterogeneous budding pattern phenotype (Zahner et al., 1996). We found that bud7/bud7 cells exhibit an axial-like phenotype and that 75% of the deletion mutants use the proximal pole for the first two divisions (Figure 2A).

View larger version (42K): [in this window] [in a new window]   Figure 2.   Budding pattern of axial-like diploid mutant strains. (A) Bud scar (Calcofluor) staining of wild-type (WT), bud7/bud7, rax2/rax2, rax1/rax1, isy1/isy1, and yor300w/yor300w cells. (B) Quantitative evaluation of first bud position in wild-type (WT) strains, axial-like mutants, and strains overexpressing Bud8. P,XXX; M, XXX; D, XXX. In all panels, >200 cells were counted.

RAX2. Chen et al. (2000) recently reported that RAX2 is required for the maintenance of the bipolar budding pattern, and their published data suggest a random budding pattern. We independently found that rax2/rax2 mutants are defective in bipolar budding, but the divisions are usually in an axial-like budding pattern (Figure 2A). Furthermore, we find that 78% of the first three budding events occurred at the proximal pole (Ni and Snyder, unpublished result). Further analysis of rax2 mutants is described below.

RAX1. Previous researchers have found that mutation of RAX1 converts the bipolar budding pattern of axl1 haploids into an axial budding pattern (Fujita et al., 1994), but the phenotype of rax1/rax1 cells has not been described. We found that homozygous rax1/rax1 cells bud in an axial-like pattern (Figure 2A); in their first two divisions they exhibit a strong bias to use the proximal pole.

ISY1 and YOR300w. Two other proteins were identified that exhibit an axial budding pattern (Figure 2A). Isy1p is a pre-mRNA-splicing factor, required for optimal splicing in vivo (Dix et al., 1999). YOR300w is an unknown gene. Quantitative analysis of the first three budding events in these mutants reveals that each possesses a bias to use the proximal pole during the first several cell cycles. yor300w/yor300w cells exhibit the most severe defect of all of the axial-like mutants; the cells often contain bud scars that span from one pole to the other (Figure 2). The analysis of YOR300w is complicated by the fact that the ORF overlaps BUD7 by 100 bp and lies immediately upstream of RAX1 (within 100 bp). Thus, the deletion removes carboxy-terminal coding sequence from BUD7 and might affect the expression of RAX1, thereby affecting two genes involved in the bipolar pattern. This combined defect might account for a phenotype that is more severe than either the bud7/bud7 or rax1/rax1 single mutants.

Random Budding Mutants

Seventy-four mutants were found to display a strong phenotype in which >50% of the cells budded randomly. An additional 38 mutants were found to exhibit a mild defect in which <50% of the cells budded randomly or sometimes random plus unipolar. The genes mutated in these strains were grouped into 11 functional categories (Table 4).

Ribosomal Proteins. The first category is ribosomal proteins. The yeast genome is predicted to encode 32 different small subunit proteins and 46 large subunit proteins. Genes for 59 of the ribosomal proteins are duplicated, resulting in 137 total ribosomal protein genes (Planta and Mager, 1998). We found that a number of the duplicated ribosomal protein genes are important for bud site selection (Table 4). rps0b/rps0b, rps7a/rps7a, rps27b/rps27b, rpl7a/rpl7a, and rpl22a/rpl22a null mutants displayed a random budding pattern and reduced growth rate (Figure 3A). rpl12b/rpl12b, rps28b/rps28b, rps29a/rps29a, rps30a/rps30a, rps1b/rps1b, and rps18b/rps18b strains exhibit a random budding pattern but grow similarly to wild-type cells; the percentage of cells exhibiting a random budding defect is reduced relative to the other strains. rpl27a/rpl27a has a reduced growth rate and also exhibits a weaker random budding defect similar to the latter group of mutants. Interestingly, strains containing deletion mutations in the homologues of the affected genes (rps0a/rps0a, rps7b/rps7b, rps27a/rps27a, rpl17b/rpl17b, rpl22b/rpl22b, rpl12a/rpl12a, rps30b/rps30b, rps1a/rps1a, rps28a/rps28a) have no obvious phenotype (i.e., budding pattern or growth rate) even though the affected proteins are often >90% identical. Thus, the duplicated copies of the ribosomal proteins are not equivalent; the copy affected in the first group is more important for the bud site selection than the copy mutated the latter group. These results suggest that inefficient translation of particular genes involved in bipolar budding affects bud site selection. Alternatively, because many of the strains have a reduced growth rate, perhaps impaired growth of these particular strains results in a loss of bipolar budding.

View larger version (74K): [in this window] [in a new window]   Figure 3.   Micrographs of random budding mutants stained with Calcofluor. A-J are examples of 10 different functional classes. (A) Ribosomal protein mutants. (B) Vesicular transport mutants. (C) Actin-cytoskeleton mutants. (D) Bud site selection and cell polarity mutants. (E) Cell wall mutants. (F) Lipid metabolism mutants. (G) Protein modification mutants. (H) Transcription factor mutants. (I) Nuclear proteins mutants. (J) Uncharacterized proteins mutants.

Vesicular Transport Proteins. Mutants for 15 proteins involved in vesicular transport exhibited a random budding pattern (Table 4). Four proteins have been implicated in exocytosis. Ypt31 is a small GTPase that belongs to the Ypt/rab family that is involved in trans-Golgi vesicle formation and localizes to sites of polarized cell growth (Jedd et al., 1997; Gerst, 1999). Bst1 was identified as a suppressor of the COPII gene SEC13, which is involved in transport from the endoplasmic reticulum to the Golgi (Elrod-Erickson and Kaiser, 1996). Sec22 and Snc2 are v-SNARE homologues involved in different steps of the secretory pathway; Sec22 is important for transport to the Golgi from the endoplasmic reticulum and Snc2 is important for late secretory steps (Woodman et al., 1996; Spang and Schekman, 1998; Gerst, 1999). Loss of SEC22 and SNC2 in homozygous diploid deletion strains result in modest bud site selection defects. Yeast haploid strains deleted for any of these four genes are not affected in the axial pattern. These results indicate that Ypt31p and Bst1p are important for the transport of membrane-associated bipolar budding components. Possible candidates are Bud8, Bud9, and/or Rax2, which are predicted transmembrane proteins.

Actin Cytoskeleton. The actin cytoskeleton is essential for both polarized growth and bud site selection (Drubin, 1990; Adams and Pringle, 1991; Adams et al., 1991; Crouzet et al., 1991; Vojtek et al., 1991; Bauer et al., 1993; Holtzman et al., 1993; Zahner et al., 1996). We identified three mutants with defects in the actin cytoskleton, rvs167/rvs167, sla1/sla1, and rvs161/rvs161, that have defects in bipolar budding similar to several act1 mutants; these had been known previously to be important for bud site selection. In addition to the cytoskeletal components that were previously shown to be important for bud site selection, we found that yeast strains mutant for two additional proteins involved in actin organization, Cap1 and Yke2, are defective in bud site selection in diploid cells (Figure 3C). Cap1 is the  subunit of actin-capping protein. It colocalizes with cortical actin patches at the site of bud emergence and at the tips of growing buds and shmoos; it does not colocalize with actin cables or with actin rings at the site of cytokinesis (Amatruda and Cooper, 1992). Careful analysis of budding patterns revealed that diploid cap1/cap1 mutants bud randomly both in the first division and in subsequent divisions (first bud: distal 29.7%; media 32.4%; proximal 29.7%; n = 200), similar to yeast bni1 mutants. Thus, in addition to its role in bipolar budding of mother cells, Cap1 might play an important role in the establishment of the distal bud tag in the daughter cell. Yke2 encodes prefoldin subunit 6, a component of the Gim protein complex that promotes formation of functional -tubulin, -tubulin, and actin (Geissler et al., 1998; Siegers et al., 1999); mutation of YKE2 causes random budding in diploids.

Bud Site Selection and Cell Polarity Proteins. Seven deletion mutants affect bud site selection and cell polarity components (Table 4; Figure 3D). Five strains lack genes known to regulate bipolar budding pattern: RSR1, BUD2, SPA2, BUD6, BNI1; mutation of these genes cause random budding either in all cell divisions or after the first division (Snyder, 1989; Ruggieri et al., 1992; Park et al., 1993; Kohno et al., 1996; Zahner et al., 1996). ROM2 and BEM4 had not been reported previously to be involved in bipolar budding. Rom2 is a GDP-GTP exchange factor for Rho1p that is activated by cell wall defects; null mutations of rom2 impair the redistribution of actin patches in response to cell wall stress (Ozaki et al., 1996; Manning et al., 1997). Similarly, Bem4 interacts with Rho-type GTPases that regulate actin cytoskeletal reorganization (Mack et al., 1996; Hirano et al., 1997). Thus, these results indicate that Rho protein regulators and interacting proteins are important for bud site selection in yeast.

Cell Wall Proteins. Six cell wall proteins were found to affect bipolar budding (Table 4). Three, fks1/fks1, gas1/gas1, and cwh8/cwh8, have a strong defect in which >50% of the cells bud randomly (Figure 3E). Fks1 is a component of -1,3-glucan synthase; -1,3-glucan is a major constituent of the yeast cell wall (Douglas et al., 1994). Gas1 is a putative glycosidase important for cell wall maintenance; it is thought to direct -1,6-glucans to cross-link to -1,3-glucan rather than to chitin (Vai et al., 1991). Cwh8 is a protein involved in the generation of the mannoprotein layer of the cell wall. Secreted proteins of cwh8 mutants have abnormally low levels of N-glycosylation (van Berkel et al., 1999). Interestingly, we found that fks1 and gas1 haploid cells do not have a defect in axial budding, whereas the cwh8 haploid strain exhibits a random budding defect.

Lipid Metabolism. We also identified a number of genes involved in lipid biosynthesis or metabolism, including SUR4, FEN1, ERG3, ERG4, and GUP1 (Figure 3F). Sur4 and Fen1 are implicated in fatty acid synthesis and post-Golgi transport; mutations in these genes allow for the bypass of V-SNARE requirement in exocytosis (Oh et al., 1997; David et al., 1998). Erg3 and Erg4 are involved in ergosterol biosynthesis (Daum et al., 1998). Gup1 is a diacylglycerol O-acyltransferase-related protein; gup1 mutants have reduced lipid synthesis and increased glycerol synthesis (Oelkers et al., 2000). Mutants lacking these genes may have altered lipid compositions that might affect the secretion or function of bipolar markers and/or cell wall components important for the bipolar pattern.

Protein Modification. We identified seven bud site selection genes whose proteins participate in protein modification (Table 4; Figure 3G). Three are involved in glycosylation. Mnn2 and Pmt2p are mannosyltransferases involved in N-linked and O-linked glycosylation, respectively (Lussier et al., 1995; Rayner and Munro, 1998). Ost3p is an oligosaccharyltransferase  subunit, which is part of a complex that transfers core oligosaccharide from dolichol carrier to Asn-X-Ser/Thr motif, and helps position the OTase (oligosaccharyltransferase) complex for efficient N-glycosylation of secretory proteins (Knauer and Lehle, 1999). These results indicate that N-linked and O-linked glycosylation play a role in bipolar budding. It is likely that these proteins affect the modification of tags or cell wall components that anchor the tags.

Transcription Factors and Chromosomal Proteins. In this screen we identified 12 mutants lacking transcriptional factors that displayed a random budding pattern phenotype (Table 4; Figure 3H). Eight, ccr4/ccr4, not5/not5, pop2/pop2, rlr1/rlr1, ctk1/ctk1, ssn6/ssn6, tup1/tup1, and hcr1/hcr1, exhibit a strong defect, and four others, sin4/sin4, ctk3/ctk3, gcr3/gcr3, and rpb4/rpb4, displayed a weak defect. Ccr4, Not5, and Pop2 are components of the CCR4 transcriptional complex, which has positive and negative effects on transcription (Draper et al., 1995; Liu et al., 1998; Oberholzer and Collart, 1998). The CCR4 complex regulates transcription during late mitosis. It may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes (Chang et al., 1999). Ssn6 and Tup1 form a complex and are general repressors of RNA polymerase II transcription (Williams et al., 1991). The Ssn6-Tup1 repressor has several distinguishing features, including its exceedingly efficient repression (>1000-fold for some target genes), the large number of genes repressed (as many as 3% of the S. cerevisiae genes), and its versatility with respect to the nature and number of activators it can prevail against (reviewed by Smith and Johnson, 2000). Because Tup1p-Ssn6 is required for the repression of many genes (including haploid-specific genes; Fujita et al., 1992; Keleher et al., 1992), deletion of Tup1-Ssn6 may induce expression of genes that affect the budding pattern. These results suggest that these transcription factors and chromosomal proteins regulate the expression of genes involved in the bipolar pattern. Ctk1 and Ctk3 are subunits of a protein kinase that phosphorylates the C-terminal domain of the large subunit of RNA polymerase II (Lee and Greenleaf, 1997). Mutation of CTK1 causes defects in both transcriptional repression and activation (Kuchin and Carlson, 1998). This kinase, which affects lexA-Tup1, might exert its activity by regulating gene expression (perhaps through Tup1) or by directly acting on components important for bud site selection. Altogether, our results indicate that gene regulation plays a very important role in bipolar bud site selection.

Nuclear Proteins. A variety of other genes involved in nuclear function or whose encoded proteins localize to the nucleus have been identified (Table 4; Figure 3I). These include SPO7, NPL3, NSR1, SFP1, NEM1, HMO1, RAI1, LSM1, TOP3, ZUO1, and TRF4. These gene products are involved in nuclear transport (Npl3, Sfp1; Blumberg and Silver, 1991; Bossie et al., 1992), have RNA-binding motifs, are involved in RNA processing/modification (Rai1, Npl3, Lsm1, Nsr1, Zuo1, and Loc1; Kondo and Inouye, 1992; Kadowaki et al., 1994; Yan et al., 1998; Costanzo et al., 2000; Tharun et al., 2000; Xue et al., 2000), are required for nuclear morphology and/or meiosis (Spo7, Nem1; Siniossoglou et al., 1998), or are DNA-binding proteins and topoisomerase (or related) homologues (Top3, Trf4, Hom1; Kim and Wang, 1992; Castano et al., 1996; Lu et al., 1996). These gene products may be involved in the expression, processing, or transport of RNA transcripts for genes involved in bipolar budding. In addition, because some of these affect processing of rRNA (Npl3, Nsr1, Rai1; Kondo and Inouye, 1992; Russell and Tollervey, 1992; Xue et al., 2000), they might function in bud site selection through ribosome biogenesis or translation.

Other Genes and Uncharacterized Genes. Other genes important for bipolar budding were also identified (Table 4; Figure 3J). These include seven known genes and 20 genes that have not been characterized previously. Deletion of 11 of the uncharacterized genes cause a strong random budding pattern, whereas the rest cause a weaker budding defect. We have named these genes BUD13 through BUD32. Further characterization of some of these genes is described below.

Many Bud Site Selection Mutants Exhibit a Defect in Cell Morphology

In addition to screening for bud site selection defects, we also examined the diploid collection for morphological defects. Wild-type diploid cells are normally ellipsoid. Previous work has demonstrated that mutants defective in apical growth, which is critical for an elongated cell shape, exhibit random budding, presumably through defective localization of tags at the poles of the cell (Sheu et al., 2000). By visually screening 3394 homozygous diploid deletion strains with the use of differential contrast interference microscopy of fixed cells, we identified 459 strains exhibiting slight to strong morphological alterations. Details of the results will be published elsewhere. Some of these shapes are described in Tables 2-4. Of particular interest, >40% of unipolar ist3/ist3 and ygl174w/ygl174w mutants exhibited enhanced growth at the end of the cell where budding occurred. These results suggest that both growth sites and division sites reside at one end of the cell in these strains.

We also noticed that 70% of our random budding pattern mutants display a round phenotype suggestive of an apical growth defect. This observation supports the hypothesis that apical growth is important for bipolar bud site selection, as suggested previously (Sheu et al., 2000).

Several Genes Affect the Budding Pattern of Haploids

We expected that mutations affecting the diploid budding pattern would be of two types. The first type would be defective in both haploid and diploid budding patterns, such as rsr1 or bud2; these mutants are thought to be defective in the general bud site selection machinery (Chant et al., 1991; Ruggieri et al., 1992; Park et al., 1993). The second would be defective in functions specifically required for the bipolar budding pattern. To determine whether any of the mutations that affected the bipolar pattern also affected the axial pattern, we analyzed the budding patterns of 103 haploid MATa deletion strains with the use of Calcofluor staining.

Of the 103 mutants, 98 appeared to display normal or near normal axial budding, suggesting that these defects were indeed specific to the bipolar budding pattern. These include genes involved actin cytoskeleton organization, vesicular trafficking, ribosomal synthesis, transcriptional factors, and most cell polarity components (Tables 2-4). However, six haploid deletion mutants bud randomly; these are rsr1, bud2, bem4, cwh8, sur4, and ygr151c (Figure 4). Rsr1 and Bud2 were previously known to be important for the budding patterns of both haploid and diploid strains (Ruggieri et al., 1992; Park et al., 1993, 1999). ORF YGR151c partially overlaps Rsr1, and thus deletion of this ORF might affect Rsr1 function, thereby accounting for the random budding phenotype. cwh8, sur4, and bem4 have not been described previously to have a bud site selection defect in haploid cells. The Sur4, Cwh8, and Bem4 proteins might therefore function with either Rsr1, Bud2, or Bud5 to help select bud sites or with the polarity establishment proteins that are thought to be targeted to bud sites by the Rsr1 machinery.

View larger version (58K): [in this window] [in a new window]   Figure 4.   Budding patterns of haploid deletion mutants. Left, bud scar staining is presented for each sample. Right, cells with more than three bud scars were scored and classified as unipolar (U) if all bud scars were concentrated at one pole, bipolar (B) if one or more scars were at each pole, and random (R) if one of more scars were in the midsection of the cell (Flescher et al., 1993). In all panels, >200 cells were counted.

Bud8-GFP Subcellular Localization in Diploid Wild-Type and Budding Mutant Strains

To further understand how the different bipolar bud site selection mutants function, we analyzed the localization of Bud8 in wild-type and many of the mutant strains. Bud8 has been proposed to act as a distal tag for bipolar bud site selection; it localizes to the bud tip (Taheri et al., 2000; Harkins, et al., 2001). As noted above, bud8/bud8 mutants are unable to bud at the distal pole and instead produce a unipolar proximal budding pattern in diploids (Zahner et al., 1996).

A high copy plasmid construct of the GFP:Bud8 gene (Harkins, et al., 2001) was transformed in a bud8/bud8 deletion strain and found to complement the bud8/ bud8 mutations (Ni and Snyder, unpublished results). When transformed into a wild-type diploid strain, the cells budded in a bipolar pattern; however, there is an increase in use of distal bud sites. This bias is especially apparent in the later budding events. For example, we found that in vegetatively growing wild-type strains containing Bud8-GFP, 95% of the second buds, and 91% of third buds, formed at distal poles, compared with only 68% of the second buds and 40% of the third buds for wild-type cells with vector alone. These results indicate that Bud8 activity is increased when overexpressed.

We next examined the localization of Bud8-GFP in the diploid wild-type strain. The Bud8-GFP was observed in ~30% of the cells. As shown in Figure 5A, Bud8-GFP localized to the presumptive bud sites in unbudded cells, the distal tips of growing buds, and the proximal pole of some daughter cells, in agreement with previous results (Harkins, et al., 2001). In addition Bud8-GFP is also localized to the mother-daughter neck in some cells and was biased toward the mother side of the neck. Double staining with Calcofluor White revealed that neck staining was observed in only the daughter cells undergoing their first division (Figure 5A). As described in DISCUSSION, we propose that Bud8 at the distal tip helps serve as a tag for the distal site, and Bud8 at the neck in new mothers might also help select these sites.

View larger version (80K): [in this window] [in a new window]   Figure 5.   Localization of Bud8-GFP in diploid wild-type (WT) and mutant strains. Cells of the indicated strains carrying the plasmid Bud8-GFP were visualized by fluorescence microscopy. (A) Wild-type strains were viewed for both GFP and Calcofluor fluorescence (same cells). (B) Bud8-GFP in vesicular transport mutants. (C) Bud8-GFP in the cwh8/cwh8 cell wall mutant. (D) Bud8-GFP in cell polarity mutants. (E) Bud8-GFP in the cap1/cap1 actin-cytoskeleton mutant. (F) Bud8-GFP in transcription factor mutants.

Bud8-GFP Localization in alg Mutants. We next examined the Bud8-GFP localization in the alg mutants, which have reduced outer chain carbohydrate modification. In alg5/alg5, alg6/alg6, alg8/alg8, and alg10/alg10 mutants, Bud8p-GFP localized to the bud tip and the mother-daughter neck in many of the cells. However, detailed quantitative analysis of the localization of the mother-daughter neck showed an important feature: more cells contained Bud8-GFP localization at the neck in alg mutants than in wild-type cells. In cells that showed any detectable GFP signal, 48.6% of wild type have Bud8-GFP staining at the neck (n = 200); in contrast, for alg5/alg5, alg6/alg6, alg8/alg8, and alg10/alg10 the percentage of cells with staining at the neck increases to 68.9, 65.8, 59.5, and 53.2% (n = 200), respectively. Thus, Bud8 outer chain modification might affect Bud8 targeting or degradation. For the latter possibility, in the absence of the outer chain modification Bud8 might be more stable and persist at the distal site longer. Alternatively, modification of other cell wall components might affect distal tag stability. Regardless, the increased presence of Bud8-GFP at the distal site (neck and bud tip) helps account for the partial unipolar budding phenotype of these cells; the cells might use the persistent Bud8 tag in multiple cell divisions.

Bud8-GFP Localization in Vesicular Transport Mutants. Nine random budding mutants in the vesicular transport category were examined for Bud8-GFP localization. bst1/bst1, vma5/vma5, vps34/vps34, vac7/ vac7, and end3/end3 each localize Bud8-GFP at polarized sites (bud tip and neck) similar to wild-type cells. Four mutants were found to have abnormal Bud8-GFP localization patterns. Clc1, the clathrin light chain, plays a role in formation of clathrin coats and clathrin-coated vesicles (Chu et al., 1996). Unlike a wild-type strain, in which >30% of the cells exhibit a polarized localization of Bud8-GFP, in clc1/clc1 mutants >90% of the mutant cells lacked detectable Bud8-GFP localization; most of the remainder have a very weak Bud8-GFP signal, often visible as a tiny spot at the bud tip (Figure 5B). This result indicates that Bud8 undergoes intracellular trafficking through a clathrin-coated vesicle pathway or that disruption of that pathway indirectly alters Bud8 localization. In another mutant, ypt31/ypt31, which lacks a small GTPase involved in trans-Golgi vesicle formation, Bud8-GFP localized in a broader patch at the tip or/and neck (Figure 5B). Two other mutants that exhibit a defect in Bud8-GFP localization are in vam3/vam3 and vam8/vam8; Vam3 and Vam8 encode syntaxin homologues (t-SNARE) thought to be important for vesicle docking and fusion reactions with the vacuole (Preston et al., 1991; Rieder and Emr, 1997; Ungermann et al., 1999). Bud8-GFP is not concentrated at the bud tip in vam3/vam3 and vam8/vam8 mutants. Instead, in vam3/vam3 mutants it localizes throughout most or all of the bud periphery (Figure 5B). In vam8/vam8 mutants, Bud8p is more diffuse at the tip and tends to form double rings at the neck (Figure 5B). These results indicate that Ypt 31, Vam3, and Vam8 may be involved in the targeting of Bud8p to the bud tip.

Bud8-GFP Localization in Cell Wall Mutants. Bud8-GFP localization was also examined in three mutants that affect cell wall function. In fks1/fks1, Bud8-GFP localization appears normal. However, in cwh8/cwh8, Bud8-GFP cannot be detected, and in gas1/gas1, Bud8 localizes to polarized sites in only 17% of the cells and was not detectable in the remainder (n = 200; Figure 5C). These results indicate that Fks1 is not required for Bud8-GFP localization, whereas Cwh8 and Gas1 are required. Thus, specific components of the cell wall are important for Bud8 localization and/or Cwh8 is important for Bud8 modification.

Bud8-GFP Localization in Budding, Cell Polarity Mutants. We also investigated Bud8-GFP localization in spa2/spa2, bud6/bud6, and bni1/bni1 mutants. Spa2, Bni1, and Pea2 are important for apical growth and have been suggested to help concentrate polarized growth and secretion at bud tips and mother-bud necks (Sheu et al., 2000). In spa2/spa2 and bud6/bud6 mutants, Bud8-GFP localization is more diffuse at the bud tip compared with wild-type cells (Figure 5D); the Bud8-GFP signal is weaker in bud6/bud6 cells than in spa2/spa2. In bni1/bni1