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Molecular and Cellular Biology, May 2001, p. 3564-3575, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3564-3575.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Localization of the Rsp5p Ubiquitin-Protein Ligase at Multiple Sites within the Endocytic Pathway

Guangli Wang,1,dagger J. Michael McCaffery,2,3 Beverly Wendland,3 Sophie Dupré,4 Rosine Haguenauer-Tsapis,4 and Jon M. Huibregtse1,*

Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712-10951; Integrated Imaging Center2 and Department of Biology,3 Johns Hopkins University, Baltimore, Maryland 21218; and Institut Jacques Monod-CNRS, Universites Paris VI and VII, 75251 Paris Cedex 05, France4

Received 12 December 2000/Returned for modification 24 January 2001/Accepted 20 February 2001

The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.

* Corresponding author. Mailing address: Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas at Austin, 2500 Speedway, Austin, TX 78712-1095. Phone: (512) 232-7700. Fax: (512) 232-3432. E-mail: huibreg{at}icmb.utexas.edu.

dagger Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014.

Molecular and Cellular Biology, May 2001, p. 3564-3575, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3564-3575.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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