SCF^Cdc4 Enables Mating Type Switching in Yeast by Cyclin-Dependent Kinase-Mediated Elimination of the Ash1 Transcriptional Repressor

Abstract

In the budding yeast Saccharomyces cerevisiae, mother cells switch mating types between a and α forms, whereas daughter cells do not. This developmental asymmetry arises because the expression of the HO endonuclease, which initiates the interconversion of a and α mating type cassettes, is extinguished by the daughter-specific Ash1 transcriptional repressor. When daughters become mothers in the subsequent cell cycle, Ash1 must be eliminated to enable a new developmental state. Here, we report that the ubiquitin ligase SCF^Cdc4 mediates the phosphorylation-dependent elimination of Ash1. The inactivation of SCF^Cdc4 stabilizes Ash1 in vivo, and consistently, Ash1 binds to and is ubiquitinated by SCF^Cdc4 in a phosphorylation-dependent manner in vitro. The mutation of a critical in vivo cyclin-dependent kinase (CDK) phosphorylation site (Thr290) on Ash1 reduces its ubiquitination and rate of degradation in vivo and decreases the frequency of mating type switching. Ash1 associates with active Cdc28 kinase in vivo and is targeted to SCF^Cdc4 in a Cdc28-dependent fashion in vivo and in vitro. Ash1 recognition by Cdc4 appears to be mediated by at least three phosphorylation sites that form two redundant diphosphorylated degrons. The phosphorylation-dependent elimination of Ash1 by the ubiquitin-proteasome system thus underpins developmental asymmetry in budding yeast.

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ACKNOWLEDGMENTS

We thank Marcia Roy and members of the Tyers laboratory for technical assistance; Kim Nasmyth, John Diffley, and Brenda Andrews for reagents; and Brenda Andrews for helpful discussions.

This work was supported by grants to C.H., F.S., and M.T. from the Canadian Institutes of Health Research (grants MOP-93571 and MOP-57795) and by grants to M.T. from the National Cancer Institute of Canada and the Wellcome Trust. T.L.B. was supported by a grant from the Biotechnology and Biological Sciences Research Council and the Engineering and Physical Sciences Research Council (BB/D019621/1). C.H. was supported by a Michael Smith Foundation for Health Research Career Investigator award and a Canadian Institutes of Health Research New Investigator award (MSH-95337), F.S. was supported by a Canada Research Chair in Structural Biology, and M.T. was supported by a Scottish Universities Life Sciences Alliance research professorship and a Royal Society Wolfson Research Merit award.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00845-10.