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Mol Cell Biol, February 1998, p. 779-789, Vol. 18, No. 2
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Role for the Ubiquitin-Proteasome System in the Vacuolar Degradation of Ste6p, the a-Factor Transporter in Saccharomyces cerevisiae

Diego Loayza and Susan Michaelis*

Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 22 August 1997/Returned for modification 6 October 1997/Accepted 29 October 1997

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.

* Corresponding author. Mailing address: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Phone: (410) 955-8286. Fax: (410) 955-4129. E-mail: susan_michaelis{at}qmail.bs.jhu.edu.




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