Analysis of the mechanisms of action of the Saccharomyces cerevisiae dominant lethal cdc42G12V and dominant negative cdc42D118A mutations.
The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42(D118A) mutant and the lethal, gain of function cdc42(G12V) mutant were examined. Cdc42(D118A,C188S)p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42(D118A) phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42(D118A) action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42(D118A, C188S)p-Cdc24p interactions and could suppress the cdc42(D118A) mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42(G12V) mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1Delta and cla4Delta cells but not ste20Delta cells, suggesting that the mechanism of cdc42(G12V) action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42(G12V) were also identified that displayed a dominant negative phenotype at 16 degrees C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.
Davis
C R
CR
Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
Richman
T J
TJ
Deliduka
S B
SB
Blaisdell
J O
JO
Collins
C C
CC
Johnson
D I
DI
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics*; D005799:Genes, Dominant; D005804:Genes, Lethal; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
D018797:Cell Cycle Proteins; D019204:GTP-Binding Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae