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J. Biol. Chem., Vol. 283, Issue 40, 27079-27088, October 3, 2008

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Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension^*

Christian M. Paumi, Matthew Chuk, Igor Chevelev, Igor Stagljar, The Stagljar group is supported by grants from the Canadian Foundation for Innovation, National Cancer Institute of Canada, Canadian Institute for Health Research, Genome Canada and the Ontario Genomics Institute, the Gebert Ruef Foundation, Novartis, and Genentech¹, and Susan Michaelis²

From the Department of Cell Biology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205 and the Terrence Donnelly Centre for Cellular and Biomolecular Research, Department of Biochemistry and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [³H]estradiol-β-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

Received for publication, April 2, 2008 , and in revised form, July 1, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM51508 (to S. M.) and National Institutes of Health Postdoctoral Award GM077024 (to C. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Depts. of Biochemistry and Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada. Tel.: 416-946-7828; Fax: 416-978-8287; E-mail: igor.stagljar{at}utoronto.ca. ² To whom correspondence may be addressed: Dept. of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-7274; Fax: 410-955-4129; E-mail: michaelis{at}jhmi.edu.

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