The Function of Two Rho Family GTPases Is Determined by Distinct Patterns of Cell Surface Localization

Abstract

Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.

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Articles of Significant Interest Selected from This Issue by the Editors

We are grateful to Brenda Temple (University of North Carolina Structural Bioinformatics Core) for assistance with structure modeling of Rho3 and Courtney Turner for technical assistance. We thank Bradley Bone (UNC Immunology Core) and Daniel Lew (Duke University) for help in the isolation and characterization of the Rho3 and Cdc42 monoclonal antibodies. We thank Guendalina Rossi for helpful discussions and critical reading of the manuscript.

This work was supported by grants from the Mathers Charitable Foundation and the National Institutes of Health (GM54712) to P. J. Brennwald.