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Mol. Cell. Biol. 29 (2): 582-601

Copyright © 2009 by the American Society for Microbiology. All rights reserved.

Nucleus-Specific and Cell Cycle-Regulated Degradation of Mitogen-Activated Protein Kinase Scaffold Protein Ste5 Contributes to the Control of Signaling Competence{triangledown}

Lindsay S. Garrenton,1,{dagger} Andreas Braunwarth,2,{ddagger} Stefan Irniger,3 Ed Hurt,2 Markus Künzler,2,§, and Jeremy Thorner1*

Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, Berkeley, California 94720-3202,1 Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universität, D-69120 Heidelberg, Germany,2 Institute of Microbiology & Genetics, Georg August University, D37077 Göttingen, Germany3

Received for publication 27 June 2008. Revision received 24 July 2008. Accepted for publication 27 October 2008.

Abstract: Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G1 phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCFCdc4 ubiquitin ligase and requires phosphorylation by the G1 cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G1 Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, Barker Hall, Room 16, Berkeley, CA 94720-3202. Phone: (510) 642-2558. Fax: (510) 642-6420. E-mail: jthorner{at}berkeley.edu

{triangledown} Published ahead of print on 10 November 2008.

{dagger} Present address: Cytokinetics, Inc., 280 East Grand Avenue, South San Francisco, CA 94080.

{ddagger} Present address: AHF Analysentechnik AG, Postfach 1543, D-72005 Tübingen, Germany.

§ Present address: Institute of Microbiology, ETH, CH-8093 Zürich, Switzerland.

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