Interactions among Rax1p, Rax2p, Bud8p, and Bud9p in Marking Cortical Sites for Bipolar Bud-site Selection in Yeast

Abstract

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.

Role of Cell Cycle-regulated Expression in the Localized Incorporation of Cell Wall Proteins in Yeast

“…We also found that haploid and diploid cwp1⌬ cells did not differ significantly from wild type in growth rate, overall cell morphology, or appearance after staining chitin with CFW (our unpublished data). The localization of Cwp1p to the birth scar suggested that it might have a role in cell division and/or in bud site selection (which involves cortical marker proteins that are localized to the cell poles; Lord et al, 2000;Harkins et al, 2001;Kang et al, 2004). However, because the percentage of budded cells with septa visible by DIC microscopy did not differ between cwp1⌬ and wild-type strains (31 and 32% for exponentially growing populations of strains LSY265 and YEF473, respectively), there seemed to be no delay in cell separation.…”

Shs1 Plays Separable Roles in Septin Organization and Cytokinesis in Saccharomyces cerevisiae

“…Those are proteins functioning in septin recruitment and ring assembly (see above), cytokinesis (see below), bud-site selection (Chant et al 1995;Sanders and Herskowitz 1996;Kang et al 2004), cell cycle control (Frenz et al 2000;Yoshida and Toh-e 2001), and mitotic signaling network (Carroll et al 1998;Longtine et al 1998Longtine et al , 2000Barral et al 1999;Lee et al 2002;. Septin serves as a scaffold for these proteins to localize and maintain at the bud neck.…”

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

“…Rax1p and Rax2p are interdependently localized and also involved in Bud9p localization via interactions (Kang et al 2004). In daughter cells of the rax2D mutant, about half of the first buds were at the distal pole, and the rest were at the Figure 3 Effect on the localization of GFP-Bud8p and GFP-Bud9p due to loss of EKC/KEOPS components.…”

“…Previous study suggests that many of the genes that specifically cause random budding, when deleted, are associated with the localization of Bud8p (Ni and Snyder 2001). Rax1p and Rax2p are interdependently related in the maintenance of bipolar budding (Kang et al 2004). The genes encoding these proteins were identified by screening genes that restored bipolar budding patterns in the haploid axl1D mutant (Fujita et al 1994).…”

“…The genes encoding these proteins were identified by screening genes that restored bipolar budding patterns in the haploid axl1D mutant (Fujita et al 1994). Bud8p and Bud9p interact with and depend on Rax1p and Rax2p, respectively, for their localization (Kang et al 2004). The delivery of Bud8p and Bud9p to the presumptive bud site and mother-bud neck, respectively, is dependent on actin.…”