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Molecular and Cellular Biology, November 2000, p. 8548-8559, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Saccharomyces cerevisiae Cdc42p GTPase Is Involved in Preventing the Recurrence of Bud Emergence during the Cell Cycle

Tamara J. Richman and Douglas I. Johnson*

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

Received 16 March 2000/Returned for modification 15 May 2000/Accepted 29 August 2000

The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an ~25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42D38Ep showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42D38E was the only mutant allele able to complement the Delta cdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42D38Ep triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 202 Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-8203. Fax: (802) 656-8749. E-mail: dijohnso{at}zoo.uvm.edu.

Molecular and Cellular Biology, November 2000, p. 8548-8559, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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