Bud8p and Bud9p, Proteins That May Mark the Sites for Bipolar Budding in Yeast

Abstract

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

“…The amplified DNA was digested with NotI and religated to create plasmid pRS316-N-NotI-MYO1, in which an in-frame NotI site had been introduced immediately after the MYO1 start codon. Finally, a NotI GFP F64L/S65T/V163A cassette (Harkins et al, 2001) was cloned into this NotI site to create plasmid pRS316-N-MYO1-GFP, which appeared to complement fully the cytokinesis and cell-separation defects of a myo1⌬ strain (unpublished data).…”

“…The Rho-family GTPase Cdc42p and its activating factor (GEF) Cdc24p are required for the polarized assembly of both the actin cytoskeleton and the septins at the beginning of the cell cycle Johnson, 1999;Pruyne and Bretscher, 2000;Cid et al, 2001;Gladfelter et al, 2001a). However, actin and the septins appear to be independent of each other for their organization at the presumptive bud site (Adams and Pringle, 1984;Ford and Pringle, 1991;Ayscough et al, 1997;Harkins et al, 2001). In the past decade, a major effort has focused on how Cdc42p controls actin organization (Johnson, 1999;Pruyne and Bretscher, 2000), but studies of the role of Cdc42p in septin organization have lagged behind.…”

“…The amplified DNA was digested with NotI and religated to create plasmid pRS316-N-NotI-MYO1, in which an in-frame NotI site had been introduced immediately after the MYO1 start codon. Finally, a NotI GFP F64L/S65T/V163A cassette (Harkins et al, 2001) was cloned into this NotI site to create plasmid pRS316-N-MYO1-GFP, which appeared to complement fully the cytokinesis and cell-separation defects of a myo1⌬ strain (unpublished data).Complete deletions of the STE20, CLA4, SKM1, and RGA2 open reading frames were constructed using the PCR method (Baudin et al, 1993;Longtine et al, 1998b). A pair of hybrid primers (Table 3) was used to amplify HIS3 (for et al, 2000).…”

Cell Polarity in Saccharomyces cerevisiae Depends on Proper Localization of the Bud9 Landmark Protein by the EKC/KEOPS Complex

“…The bud sites during axial budding are marked with the axial landmarks Axl1p, Axl2p, Bud3p, and Bud4p (Fujita et al 1994;Halme et al 1996;Sanders and Herskowitz 1996). The bipolar landmarks Bud8p and Bud9p of the distal and proximal poles, respectively, determine the polarization axis of budding in diploid cells (Harkins et al 2001). These cortical landmarks provide recognition sites for the Rsr1p/Bud2p/Bud5p GTPase signaling module (Kang et al 2001;Park and Bi 2007).…”

“…BUD8 and BUD9 were originally identified as specific genes essential for normal bipolar budding. The bud8D and bud9D mutants bud at the proximal and distal poles, respectively (Harkins et al 2001). In addition, Bud8p and Bud9p are localized at the distal and proximal poles, respectively (Harkins et al 2001).…”