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Vol. 15, Issue 7, 3031-3041, July 2004

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Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body

Anne Eugster ^*, Eve-Isabelle Pécheur , Fabrice Michel , Barbara Winsor , François Letourneur ^*, and Sylvie Friant  ^||

^* Laboratoire de Transport et Compartimentation Intracellulaire; Laboratoire de RMN et Bioinformatique Structurales; Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR5086 Centre National de la Recherche Scientifique, Institut Fédératif de Recherche 128 BioSciences Lyon-Gerland, 69367 Lyon, France; and Laboratoire Modèles Levure de Pathologies Humaines, IBMC, FRE2375 Centre National de la Recherche Scientifique, 67084 Strasbourg, France

Submitted November 7, 2003; Revised March 11, 2004; Accepted April 9, 2004
Monitoring Editor: Jennifer Lippincott-Schwartz

At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P₂ and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P₂ via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P₂- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.

Abbreviations used: ALP, alkaline phosphatase; CPY, carboxypeptidase Y; ENTH, epsin NH₂-terminal homology; ESCRT, endosomal sorting complex required for transport; FYVE domain, Fab1, YGL023, Vps27 and EEA1 domain; MVB, multivesicular body; PC, phosphatidylcholine; PtdIns(3,5)P₂, phosphatidylinositol 3,5-bisphosphate; Ub, ubiquitin; UIM, Ub-interacting motif; Vps, vacuolar protein sorting.

^|| Corresponding author. E-mail address: S.Friant{at}ibmc.u-strasbg.fr.

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