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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.
Highlights
From the ‡Department of Molecular Genetics and §Graduate Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, Ohio 43210-1292
We show that localization of Cdc24, a guanine nucleotide exchange factor for Cdc42, to the proper bud site is dependent on Rsr1
green fluorescent protein (GFP)-Rsr1 appeared to be evenly distributed on the plasma membrane in over 50% of unbudded cells (n ϭ 145, 1) and was enriched in a patch at the incipient bud site (20%; Fig. 1A, 2, 3, and 8)
Summary
From the ‡Department of Molecular Genetics and §Graduate Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, Ohio 43210-1292 Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The lysine repeat and the CAAX motif of Rsr1/Bud are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud is essential for its function in proper bud site selection because both mutations resulted in random bud site selection. The GTPase module consisting of Rsr1/Bud (Rsr hereafter), its GDP-GTP exchange factor Bud, and its GTPase-activating protein Bud is essential for selecting the proper site for polarized growth in both haploid and diploid cells (4 –7). We show that localization of Cdc, a guanine nucleotide exchange factor for Cdc, to the proper bud site is dependent on Rsr
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