A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae

Abstract

A genome-wide screen of 4168 homozygous diploid yeast deletion strains has been performed to identify nonessential genes that participate in the bipolar budding pattern. By examining bud scar patterns representing the sites of previous cell divisions, 127 mutants representing three different phenotypes were found: unipolar, axial-like, and random. From this screen, 11 functional classes of known genes were identified, including those involved in actin-cytoskeleton organization, general bud site selection, cell polarity, vesicular transport, cell wall synthesis, protein modification, transcription, nuclear function, translation, and other functions. Four characterized genes that were not known previously to participate in bud site selection were also found to be important for the haploid axial budding pattern. In addition to known genes, we found 22 novel genes (20 are designated BUD13-BUD32) important for bud site selection. Deletion of one resulted in unipolar budding exclusively from the proximal pole, suggesting that this gene plays an important role in diploid distal budding. Mutations in 20 other novel BUD genes produced a random budding phenotype and one produced an axial-like budding defect. Several of the novel Bud proteins were fused to green fluorescence protein; two proteins were found to localize to sites of polarized cell growth (i.e., the bud tip in small budded cells and the neck in cells undergoing cytokinesis), similar to that postulated for the bipolar signals and proteins that target cell division site tags to their proper location in the cell. Four others localized to the nucleus, suggesting that they play a role in gene expression. The bipolar distal marker Bud8 was localized in a number of mutants; many showed an altered Bud8-green fluorescence protein localization pattern. Through the genome-wide identification and analysis of different mutants involved in bipolar bud site selection, an integrated pathway for this process is presented in which proximal and distal bud site selection tags are synthesized and localized at their appropriate poles, thereby directing growth at those sites. Genome-wide screens of defined collections of mutants hold significant promise for dissecting many biological processes in yeast.

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.

Figure 2

Budding pattern of axial-like diploid mutant strains. (A) Bud scar (Calcofluor) staining of wild-type (WT), bud7Δ/bud7Δ, rax2Δ/rax2Δ, rax1Δ/rax1Δ, isy1Δ/isy1Δ, and yor300wΔ/yor300wΔ 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.

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.

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.

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 cwh8Δ/cwh8Δ cell wall mutant. (D) Bud8-GFP in cell polarity mutants. (E) Bud8-GFP in the cap1Δ/cap1Δ actin-cytoskeleton mutant. (F) Bud8-GFP in transcription factor mutants.

Figure 6

Localization of Bud-GFP proteins. (A) Bud14/Yar014c-GFP, which is localized as a patch at the presumptive bud sites in unbudded cells, the distal tips of growing buds and the mother-daughter bud neck in large-budded cells. (B) Bud15/Ybl047c-GFP, which at the bud tip in small and large budded cells and at the mother-daughter neck in cells undergoing cytokinesis. (C) Six other GFP fusion proteins localized to nucleus.

Figure 7

A model for bipolar bud site selection that integrates the results of this study. As described in the text the different bud site selection proteins are likely to act at the sites indicated in the diagram. The bud genes indicated with a asterisk might either participate in transcription or other nuclear processes. ER, endoplasmic reticulum. mRNP, mRNA-protein; Pr, proteins; PVC, prevacuolar compartment.

Figure 8

An improved model of Bud8 localization. Bud8, indicated by the gray-shaded areas, might help direct new growth sites at the distal tip during both the first and subsequent divisions. See DISCUSSION for more details. M, mother; D, daughter.