Molecular Biology of the Cell Vol. 15, 3031–3041, July 2004 Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body Anne Eugster,* Eve-Isabelle Pe ´cheur, † Fabrice Michel, ‡ Barbara Winsor, § Franc ¸ois 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 Prote ´ines, UMR5086 Centre National de la Recherche Scientifique, Institut Fe ´de ´ratif de Recherche 128 BioSciences Lyon-Gerland, 69367 Lyon, France; and § Laboratoire Mode `les 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)P 2 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)P 2 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)P 2 - and the Vps27p-ubiquitin– driven sorting machinery at the multivesicular body. INTRODUCTION Endosomes are crossroads between the biosynthetic and the endocytic pathways. Here, proteins destined for the lyso- some/vacuole are segregated away from proteins recycling back to the plasma membrane or to the Golgi apparatus. A critical membrane segregation and protein-sorting event takes place in late endosomes, regions of endosomal mem- branes invaginate and vesicles bud into the lumen of the organelle, giving rise to the multivesicular body (MVB; Felder et al., 1990). Mature MVB fuses with the vacuole/ lysosome releasing internal vesicles into its lumen and thereby exposing them to the activity of hydrolytic enzymes (Futter et al., 1996; Odorizzi et al., 1998). This mechanism allows a subset of endosomal membrane proteins to be sorted to the lumen of the lysosome/vacuole, whereas oth- ers are either selectively recycled back to the Golgi or to the plasma membrane, or they end up on the lysosomal/vacu- olar membrane (Pelham, 2002). In yeast, the MVB pathway is crucial for the regulated turnover of pheromone receptors and of some permeases (Odorizzi et al., 1998). Further, bio- synthetic transmembrane proteins such as carboxypeptidase S (Cps1p) and Phm5p follow this route to reach their final destination, the vacuolar lumen (Odorizzi et al., 1998; Reg- giori and Pelham, 2001; Epple et al., 2003). Ubiquitination has been implicated as an endosomal-sort- ing signal. Cps1p and Phm5p ubiquitination is essential to target them into the vacuolar lumen after sorting at the MVB (Katzmann et al., 2001; Reggiori and Pelham, 2001). Potential cargo receptors responsible for recognition and recruitment of ubiquitinated cargo into MVB vesicles are class E Vps (vacuolar protein sorting) proteins. Class E mutants accu- mulate cargo destined for the vacuole in an exaggerated endosomal structure and all (currently identified) class E proteins are required for sorting at the MVB (Conibear and Stevens, 1998). Vps23p, a class E protein of the ESCRT-I complex (endosomal-sorting complex required for trans- port), carries an ubiquitin-conjugating (UBC)-like domain and interacts directly with ubiquitin in vitro and with ubi- quitinated Cps1p in vivo (Katzmann et al., 2001). By binding to ubiquitinated cargo and then activating ESCRT-II, which in turn acts upstream of ESCRT-III, ESCRT-I is the first effector of a coordinated cascade necessary for ubiquitin- dependent protein sorting at the MVB (Babst et al., 2002a, 2002b). Two other class E proteins, Vps27p and Hse1p, contain ubiquitin-interacting motifs (UIMs) and bind ubiq- uitin in vitro (Bilodeau et al., 2002; Polo et al., 2002; Raiborg et al., 2002; Shih et al., 2002). Mutations in the UIMs of Vps27p and Hse1p cause defects in sorting of Cps1p and Ste2p, suggesting that a Vps27p/Hse1p complex is a sorting receptor for ubiquitinated proteins at the MVB. Vps27p binds PtdIns(3)P via its FYVE domain (Burd and Emr, 1998; Gary et al., 1998; Gaullier et al., 1998; Wishart et al., 2001; Burda et al., 2002). PtdIns(3)P is found on the cytoplasmic Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E03–11– 0793. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03–11– 0793.  Corresponding author. E-mail address: S.Friant@ibmc.u-strasbg.fr. Abbreviations used: ALP, alkaline phosphatase; CPY, car- boxypeptidase Y; ENTH, epsin NH 2 -terminal homology; ES- CRT, endosomal sorting complex required for transport; FYVE domain, Fab1, YGL023, Vps27 and EEA1 domain; MVB, mul- tivesicular body; PC, phosphatidylcholine; PtdIns(3,5)P 2 , phos- phatidylinositol 3,5-bisphosphate; Ub, ubiquitin; UIM, Ub-inter- acting motif; Vps, vacuolar protein sorting. © 2004 by The American Society for Cell Biology 3031