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Eukaryotic Cell, June 2002, p. 458-468, Vol. 1, No. 3
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.3.458-468.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Saccharomyces cerevisiae Cdc42p Localizes to Cellular Membranes and Clusters at Sites of Polarized Growth

Tamara J. Richman, Mathew M. Sawyer, and Douglas I. Johnson*

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

Received 13 December 2001/ Accepted 11 March 2002

The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.

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

Eukaryotic Cell, June 2002, p. 458-468, Vol. 1, No. 3
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.3.458-468.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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