Previous Article | Next Article 
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,
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.

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.
This article has been cited by other articles:
-
Strochlic, T. I., Schmiedekamp, B. C., Lee, J., Katzmann, D. J., Burd, C. G.
(2008). Opposing Activities of the Snx3-Retromer Complex and ESCRT Proteins Mediate Regulated Cargo Sorting at a Common Endosome. Mol. Biol. Cell
19: 4694-4706
[Abstract]
[Full Text]
-
Leon, S., Erpapazoglou, Z., Haguenauer-Tsapis, R.
(2008). Ear1p and Ssh4p Are New Adaptors of the Ubiquitin Ligase Rsp5p for Cargo Ubiquitylation and Sorting at Multivesicular Bodies. Mol. Biol. Cell
19: 2379-2388
[Abstract]
[Full Text]
-
Estrella, L. A., Krishnamurthy, S., Timme, C. R., Hampsey, M.
(2008). The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae. J. Biol. Chem.
283: 5327-5334
[Abstract]
[Full Text]
-
Nikko, E., Andre, B.
(2007). Split-Ubiquitin Two-Hybrid Assay To Analyze Protein-Protein Interactions at the Endosome: Application to Saccharomyces cerevisiae Bro1 Interacting with ESCRT Complexes, the Doa4 Ubiquitin Hydrolase, and the Rsp5 Ubiquitin Ligase. Eukaryot Cell
6: 1266-1277
[Abstract]
[Full Text]
-
Watson, H., Bonifacino, J. S.
(2007). Direct Binding to Rsp5p Regulates Ubiquitination-independent Vacuolar Transport of Sna3p. Mol. Biol. Cell
18: 1781-1789
[Abstract]
[Full Text]
-
McNatt, M. W., McKittrick, I., West, M., Odorizzi, G.
(2007). Direct Binding to Rsp5 Mediates Ubiquitin-independent Sorting of Sna3 via the Multivesicular Body Pathway. Mol. Biol. Cell
18: 697-706
[Abstract]
[Full Text]
-
Ing, B., Shteiman-Kotler, A., Castelli, M., Henry, P., Pak, Y., Stewart, B., Boulianne, G. L., Rotin, D.
(2007). Regulation of Commissureless by the Ubiquitin Ligase DNedd4 Is Required for Neuromuscular Synaptogenesis in Drosophila melanogaster. Mol. Cell. Biol.
27: 481-496
[Abstract]
[Full Text]
-
Ren, J., Kee, Y., Huibregtse, J. M., Piper, R. C.
(2007). Hse1, a Component of the Yeast Hrs-STAM Ubiquitin-sorting Complex, Associates with Ubiquitin Peptidases and a Ligase to Control Sorting Efficiency into Multivesicular Bodies. Mol. Biol. Cell
18: 324-335
[Abstract]
[Full Text]
-
Staub, O., Rotin, D.
(2006). Role of Ubiquitylation in Cellular Membrane Transport. Physiol. Rev.
86: 669-707
[Abstract]
[Full Text]
-
Arnason, T. G., Pisclevich, M. G., Dash, M. D., Davies, G. F., Harkness, T. A. A.
(2005). Novel Interaction between Apc5p and Rsp5p in an Intracellular Signaling Pathway in Saccharomyces cerevisiae. Eukaryot Cell
4: 134-146
[Abstract]
[Full Text]
-
Heidecker, G., Lloyd, P. A., Fox, K., Nagashima, K., Derse, D.
(2004). Late Assembly Motifs of Human T-Cell Leukemia Virus Type 1 and Their Relative Roles in Particle Release. J. Virol.
78: 6636-6648
[Abstract]
[Full Text]
-
Dunn, R., Klos, D. A., Adler, A. S., Hicke, L.
(2004). The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo. JCB
165: 135-144
[Abstract]
[Full Text]
-
Murray, R. Z., Jolly, L. A., Wood, S. A.
(2004). The FAM Deubiquitylating Enzyme Localizes to Multiple Points of Protein Trafficking in Epithelia, where It Associates with E-cadherin and {beta}-catenin. Mol. Biol. Cell
15: 1591-1599
[Abstract]
[Full Text]
-
Angers, A., Ramjaun, A. R., McPherson, P. S.
(2004). The HECT Domain Ligase Itch Ubiquitinates Endophilin and Localizes to the trans-Golgi Network and Endosomal System. J. Biol. Chem.
279: 11471-11479
[Abstract]
[Full Text]
-
Care, A., Vousden, K. A., Binley, K. M., Radcliffe, P., Trevethick, J., Mannazzu, I., Sudbery, P. E.
(2004). A Synthetic Lethal Screen Identifies a Role for the Cortical Actin Patch/Endocytosis Complex in the Response to Nutrient Deprivation in Saccharomyces cerevisiae. Genetics
166: 707-719
[Abstract]
[Full Text]
-
Katzmann, D. J., Sarkar, S., Chu, T., Audhya, A., Emr, S. D.
(2004). Multivesicular Body Sorting: Ubiquitin Ligase Rsp5 Is Required for the Modification and Sorting of Carboxypeptidase S. Mol. Biol. Cell
15: 468-480
[Abstract]
[Full Text]
-
Blondel, M.-O., Morvan, J., Dupre, S., Urban-Grimal, D., Haguenauer-Tsapis, R., Volland, C.
(2004). Direct Sorting of the Yeast Uracil Permease to the Endosomal System Is Controlled by Uracil Binding and Rsp5p-dependent Ubiquitylation. Mol. Biol. Cell
15: 883-895
[Abstract]
[Full Text]
-
Bouamr, F., Melillo, J. A., Wang, M. Q., Nagashima, K., de Los Santos, M., Rein, A., Goff, S. P.
(2003). PPPYEPTAP Motif Is the Late Domain of Human T-Cell Leukemia Virus Type 1 Gag and Mediates Its Functional Interaction with Cellular Proteins Nedd4 and Tsg101. J. Virol.
77: 11882-11895
[Abstract]
[Full Text]
-
Meriin, A. B., Zhang, X., Miliaras, N. B., Kazantsev, A., Chernoff, Y. O., McCaffery, J. M., Wendland, B., Sherman, M. Y.
(2003). Aggregation of Expanded Polyglutamine Domain in Yeast Leads to Defects in Endocytosis. Mol. Cell. Biol.
23: 7554-7565
[Abstract]
[Full Text]
-
Abe, F., Iida, H.
(2003). Pressure-Induced Differential Regulation of the Two Tryptophan Permeases Tat1 and Tat2 by Ubiquitin Ligase Rsp5 and Its Binding Proteins, Bul1 and Bul2. Mol. Cell. Biol.
23: 7566-7584
[Abstract]
[Full Text]
-
Umebayashi, K., Nakano, A.
(2003). Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane. JCB
161: 1117-1131
[Abstract]
[Full Text]
-
Davies, B. A., Topp, J. D., Sfeir, A. J., Katzmann, D. J., Carney, D. S., Tall, G. G., Friedberg, A. S., Deng, L., Chen, Z., Horazdovsky, B. F.
(2003). Vps9p CUE Domain Ubiquitin Binding Is Required for Efficient Endocytic Protein Traffic. J. Biol. Chem.
278: 19826-19833
[Abstract]
[Full Text]
-
Wedaman, K. P., Reinke, A., Anderson, S., Yates, J. III, McCaffery, J. M., Powers, T.
(2003). Tor Kinases Are in Distinct Membrane-associated Protein Complexes in Saccharomyces cerevisiae. Mol. Biol. Cell
14: 1204-1220
[Abstract]
[Full Text]
-
Kaminska, J., Gajewska, B., Hopper, A. K., Zoladek, T.
(2002). Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae. Mol. Cell. Biol.
22: 6946-6948
[Abstract]
[Full Text]
-
Harkness, T. A. A., Davies, G. F., Ramaswamy, V., Arnason, T. G.
(2002). The Ubiquitin-Dependent Targeting Pathway in Saccharomyces cerevisiae Plays a Critical Role in Multiple Chromatin Assembly Regulatory Steps. Genetics
162: 615-632
[Abstract]
[Full Text]
-
Shcherbik, N., Kumar, S., Haines, D. S.
(2002). Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1. J. Cell Sci.
115: 1041-1048
[Abstract]
[Full Text]
-
Harty, R. N., Brown, M. E., McGettigan, J. P., Wang, G., Jayakar, H. R., Huibregtse, J. M., Whitt, M. A., Schnell, M. J.
(2001). Rhabdoviruses and the Cellular Ubiquitin-Proteasome System: a Budding Interaction. J. Virol.
75: 10623-10629
[Abstract]
[Full Text]
-
Dupre, S., Haguenauer-Tsapis, R.
(2001). Deubiquitination Step in the Endocytic Pathway of Yeast Plasma Membrane Proteins: Crucial Role of Doa4p Ubiquitin Isopeptidase. Mol. Cell. Biol.
21: 4482-4494
[Abstract]
[Full Text]
-
Dunn, R., Hicke, L.
(2001). Multiple Roles for Rsp5p-dependent Ubiquitination at the Internalization Step of Endocytosis. J. Biol. Chem.
276: 25974-25981
[Abstract]
[Full Text]