Figure 6.5 Bud site selection in Saccharomyces cerevisiae. The pattern of budding is characterized by the orientation of the bud scars with respect to the birth scar. In calcofluor staining, the birth scars appear as unstained areas whereas the birth scars are brightly fluorescent. In axial pattern the bud scars always lie juxtaposed to the birth scar and to each other forming a continuous line of scars. In bipolar budding, the bud scars are seen opposite to the birth scar and also adjacent to it as shown in the figure. In random pattern, the bud scars are seen arranged randomly on the yeast cell.
selected by observing microcolonies formed after the mother cell has undergone one to three cell divisions under a microscope. The pattern of budding is revealed by the arrangement of the daughter cells with respect to the mother cell in each microcolony. The original genetic screen identified four genes (BUD1, BUD2, BUD3 and BUD4) required for the specification of the axial budding pattern. A fifth gene BUD5 was identified by molecular approaches (Chant et al., 1991). Analysis of the function of the genes in haploids and diploids revealed that BUD1, BUD2 and BUD5 are required by both types of cells to select bud sites at correct positions, whereas mutation in BUD3 and BUD4 affected only haploid cells. Further work from various laboratories identified other genes that distinctly affected bud site selection in haploids and diploids. The mechanism of bud-site selection follows a two-step process. First, the yeast cell integrates intrinsic spatial information to define the site for the growth of the new bud. Proteins that persist at the site of the previous bud from one cell division to the next produce these spatial cues. Localization experiments using GFP-tagged proteins support the persistence of BUD3, BUD4 and BUD10 at previous bud sites after each division cycle. Once the site is selected, the next step involves recruitment of a GTP-binding protein BUD1 and its regulators BUD2 and BUD5 to the site previously marked by BUD3, BUD4 and BUD10, leading to the localized activation of BUD1. It is believed that active BUD1 activates CDC42 locally by interacting with its GTP-exchange factor CDC24. Active CDC42 then induces actin cytoskeletal reorganization by regulating the activity of actin-binding proteins. As a result of cell polarization, the protein transport machinery delivers membranes and components of cell wall biosynthesis at the site of the growing bud.
Bud site selection in diploids is mediated by a different group of landmark proteins BUD8 and BUD9 that mark the poles of the cell by recruiting RAX2p, which persists at the poles for many generations and is believed to activate CDC42p via BUDlp. In the absence of BUD8 or BUD9, RAX2p fails to localize correctly, causing the diploid to bud in a random pattern. A schematic representation of the molecular machinery that guides the axis of polarization during budding is shown in Figure 6.6.
Although it may appear that generation of polarity in unicellular yeast is different from that in epithelial cells of multicellular organisms, the basic core mechanism of
Cdc 24 GEF
Rga 1 Rga 2 Bem 3 GAPS
Ste 20 Gln 4
Glc 1 Glc 2
Actin microtubules septins
Figure 6.6 Mechanisms that generate cell polarity during bud-site selection in S. cerevisiae. The spatial landmark proteins on the cell surface guide the cellular machinery to build the axis of polarization through the GTP-binding proteins BUD1 and CDC42. Reproduced with permission Figure 4a from Chant, J. (1999), Ann. Rev. Cell Dev. Biol. 15, 365-391. © Annual Reviews Inc.
organizing the cytoskeleton using CDC42 and actin-binding proteins arose very early during evolution to be used universally by all eukaryotic organisms.
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