Ubiquitination Mediated by the Npi1p/Rsp5p Ubiquitin-protein Ligase Is Required for Endocytosis of the Yeast Uracil Permease

doi:10.1074/jbc.271.18.10946

Ubiquitination Mediated by the Npi1p/Rsp5p Ubiquitin-protein Ligase Is Required for Endocytosis of the Yeast Uracil Permease (*)

From the (1)Institut Jacques Monod/CNRS, Université Paris7-Denis Diderot, 2, place Jussieu, 75251 Paris Cedex 05, France,
(2)Institut de Biologie Moleculaire et Cellulaire/CNRS, 15, rue Descartes, 67084 Strasbourg, France, and
(3)Laboratoire de Physiologie Cellulaire et de Génétique des Levures, Université Libre de Bruxelles, Campus plaine CP 244, Boulevard du Triomphe, B-1050 Bruxelles, Belgium

^§ To whom correspondence should be addressed. Tel. 33-1-44-27-63-86; Fax: 33-1-44-27-59-94; rht{at}ccr.jussieu.fr.

Abstract

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.

Footnotes

↵* This work was supported by grants from CNRS, the University Paris7-Denis Diderot, the Fondation de la Recherche Médicale, and the Centre des Comités de Lutte contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Ub

ubiquitin

HA

hemagglutinin

Tricine

N-tris(hydroxymethyl)methylglycine

β-gal

β-galactosidase

PAGE

polyacrylamide gel electrophoresis.
↵²T. Zoladek, personal communication.
- Received December 21, 1995.
- Revision received January 31, 1996.