Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

doi:10.1074/jbc.273.2.849

Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42^G12V and Dominant Negative cdc42^D118A Mutations*

From the Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405

Abstract

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 ^D118Amutant and the lethal, gain of functioncdc42 ^G12V mutant were examined. Cdc42^D118A,C188Sp 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 ofcdc42 ^D118A action. Five cdc42mutations 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,C188Sp-Cdc24p interactions and could suppress the cdc42 ^D118A mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. Thecdc42 ^G12V mutant phenotypes were suppressed by the intragenic T35A and K183–187Q mutations and in skm1Δ and cla4Δ cells but not ste20Δ cells, suggesting that the mechanism of cdc42 ^G12Vaction is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors ofcdc42 ^G12V were also identified that displayed a dominant negative phenotype at 16 °C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.

Footnotes

↵* This research was supported by National Science Foundation Grant DMB-9405972, a grant from the Lucille P. Markey Charitable Trust, and a UVM-HELiX (Howard Hughes Program for Training in Biology) fellowship (to J. O. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Present address: Biology Dept., Eastern Nazarene College, 23 E. Elm Ave., Quincy, MA 02170.
↵§ To whom correspondence should be addressed: Dept. of Microbiology & Molecular Genetics, University of Vermont, 202A Stafford Hall, Burlington, VT 05405. Tel.: 802-656-8203; Fax: 802-656-8749; E-mail:dijohnso{at}zoo.uvm.edu.
↵1 The abbreviations used are: PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoside.
↵2 P. Miller and D. I. Johnson, unpublished results.
↵3 T. J. Richman and D. I. Johnson, manuscript in preparation.
↵4 M. Ziman and D. I. Johnson, unpublished results.
- Received July 25, 1997.
- Revision received September 29, 1997.
The American Society for Biochemistry and Molecular Biology, Inc.