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J. Biol. Chem., Vol. 283, Issue 9, 5327-5334, February 29, 2008

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The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae^*

From the Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho^– phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4 mutant in the presence of limiting inorganic phosphate (P_i). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P_i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P_i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho⁺ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P_i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.

Received for publication, May 2, 2007 , and in revised form, November 28, 2007.

^* This work was supported by National Institutes of Health Initiative for Maximizing Student Diversity Award-UMDNJ/Rutgers University Pipeline Program R25 GM55145 (to M. J. Leibowitz), National Institutes of Health Graduate Training in Cellular and Molecular Biology Grant T32 GM08360 (to K. Madura), by National Institutes of Health Grant F31 GM67388 (to L. A. E.), and by National Institutes of Health Grant RO1 GM39484 (to M. H.). 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 Present address: Dept. of Biology, University of Puerto Rico-Rio Piedras, San Juan, Puerto Rico 00936.

² These authors contributed equally to this work.

³ Present address: Dept. of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612.

⁴ To whom correspondence should be addressed: Dept. of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 683 Hoes Lane West, Piscataway, NJ 08854. Tel.: 732-235-5888; Fax: 732-235-5889; E-mail: michael.hampsey{at}umdnj.edu.

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