Abstract
Site-specific activation of the Rho-type GTPase Cdc42p by its guanine-nucleotide exchange factor (GEF) Cdc24p is critical for the establishment of cell polarity. Here we show that binding of Cdc24p to the small GTPase Rsr1p/Bud1p is required for its recruitment to the incipient bud site. Rsr1p/Bud1p binds to the CH-domain of Cdc24p, which is essential for its function in vivo. We have identified a cdc24-mutant allele, which is specifically defective for bud-site selection. Our results suggest that Cdc24p is auto-inhibited by an intramolecular interaction with its carboxy-terminal PB1-domain. Rsr1p/Bud1p appears to activate the GEF activity of Cdc24p in vivo, possibly by triggering a conformational change that dissociates the PB1-domain from its intramolecular binding site. Genetic experiments suggest that Bem1p functions as a positive regulator of Cdc24p by binding to the PB1-domain of Cdc24p, thereby preventing its re-binding to the intramolecular inhibitory site. Taken together, our results support a two-step molecular mechanism for the site-specific activation of Cdc24p, which involves Rsr1p/Bud1p and the adaptor protein Bem1p.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Adaptor Proteins, Signal Transducing / metabolism
-
Alleles
-
Cell Cycle Proteins / antagonists & inhibitors*
-
Cell Cycle Proteins / chemistry
-
Cell Cycle Proteins / genetics
-
Cell Cycle Proteins / metabolism*
-
Cell Membrane / metabolism
-
Gene Expression Regulation, Fungal / genetics*
-
Guanine Nucleotide Exchange Factors / antagonists & inhibitors*
-
Guanine Nucleotide Exchange Factors / chemistry
-
Guanine Nucleotide Exchange Factors / genetics
-
Guanine Nucleotide Exchange Factors / metabolism*
-
Mutation / genetics
-
Protein Binding
-
Protein Structure, Tertiary
-
Protein Transport
-
Saccharomyces cerevisiae / cytology
-
Saccharomyces cerevisiae / genetics
-
Saccharomyces cerevisiae / metabolism*
-
Saccharomyces cerevisiae Proteins / antagonists & inhibitors*
-
Saccharomyces cerevisiae Proteins / chemistry
-
Saccharomyces cerevisiae Proteins / genetics
-
Saccharomyces cerevisiae Proteins / metabolism*
-
rab GTP-Binding Proteins / genetics
-
rab GTP-Binding Proteins / metabolism
Substances
-
Adaptor Proteins, Signal Transducing
-
CDC24 protein, S cerevisiae
-
Cell Cycle Proteins
-
Guanine Nucleotide Exchange Factors
-
Saccharomyces cerevisiae Proteins
-
BEM1 protein, S cerevisiae
-
RSR1 protein, S cerevisiae
-
rab GTP-Binding Proteins