Localization of the Rsr1/Bud1 GTPase Involved in Selection of a Proper Growth Site in Yeast

Abstract

Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/ Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.Cells of the budding yeast Saccharomyces cerevisiae undergo oriented cell division by selecting a specific site for polarized growth on their cell cortex (1-3). Haploid a and ␣ cells bud in an axial pattern, whereas diploid a/␣ cells bud in a bipolar pattern. The GTPase module consisting of Rsr1/Bud1 (Rsr1 hereafter), its GDP-GTP exchange factor Bud5, and its GTPase-activating protein Bud2 is essential for selecting the proper site for polarized growth in both haploid and diploid cells (4 -7). Based on genetic and biochemical data, we proposed previously that the Rsr1 GTPase module directs bud site assembly to occur at specific locations by recruiting components such as Cdc24 required for bud formation to that site (8). Recruiting these proteins to the presumptive bud site is thought to direct the cytoskeleton and secretory apparatus toward the bud site, thereby restricting new growth to the bud (9). One of the key questions in understanding the molecular basis of cell polarity is how specific sites for actin polymerization are determined.To understand the mechanism of action of the Rsr1 GTPase module, it is crucial to determine whether any of its components are localized to the presumptive bud site. We reported previously that both Bud2 and Bud5 are localized to the presumptive bud site and to discrete sites during the cell cycle (10, 11). This localization is essential for selection of a specific site for growth. Here we report that Rsr1 is broadly distributed on the plasma membrane and is highly concentrated at the incipient bud site and polarized growth sites. Mutational studies indicated that the lysine repeat in the hypervariable region and the CAAX motif of Rsr1 are important for its localization and its role in selection of a proper site for growth. We also show that localization of Cdc24, a guanine nucleotide exchange factor for Cdc42, to the proper bud site is dependent on Rsr1. EXPERIMENTAL PR...

Rap1: a key regulator in cell-cell junction formation

“…In this respect, the analogy with yeast is striking; the yeast ortholog of Rap1, Rsr1, in response to positional cues is responsible for guiding the position of the future bud. Activation of Rsr1 results in the association with a GEF for Cdc42 and the subsequent activation of Cdc42 as a regulator of the actin cytoskeleton (Chant and Herskowitz, 1991;Park et al, 2002). Despite this analogy, the function of Rap1 in mammalian cells seems to be more complex and not restricted to the regulation of the actin cytoskeleton.…”

Interaction between bud-site selection and polarity-establishment machineries in budding yeast

“…Moreover, in Candida Bud5 is localized to hyphal tips [45]. Similarly, in S. cerevisiae, Rsr1 and Bud5 become concentrated at the polarity site during bud emergence [22,23,25]. In addition, RSR1 overexpression was found to partly suppress the temperature sensitivity of specific cdc42 alleles [46], suggesting that Rsr1 can assist in polarity establishment per se, in addition to influencing the position of the polarity site.…”

“…The landmark proteins interact with Bud5, a GEF that promotes localized activation of Rsr1 [21][22][23][24][25][26] (figure 2d). (a) A random site on the cell membrane exhibits a stochastic increase in GTPCdc42.…”

A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous FungusAshbya gossypii

“…In S. cerevisiae, Rsr1p/Bud1p localizes to sites of growth in a cell cycle-dependent manner (Park et al, 2002). This is of peculiar interest because it points to a more important role than simple bud-site selection at the G1/S transition.…”