Wide-type CHO and mutant ldlF cell line were obtained from M

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Wide-type CHO and mutant ldlF cell line were obtained from M. and COP are not found on endosomes. However, COP, which is normally present on endosomes, is usually no longer recruited when COP is usually missing. In contrast, all COP subunits, except obviously COP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that this biogenesis of multivesicular endosomes is usually coupled to early endosome business and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas , , and COP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of and/or COP. After internalization, cell surface proteins and lipids, as well as solutes, first appear in peripheral early endosomes. Depending on their fate, internalized molecules can then either be recycled back to the cell surface for reutilization or transported to late endosomes and then lysosomes for degradation (Gruenberg and Maxfield, 1995; Mellman, 1996). These two pathways exhibit major differences with respect to membrane business and dynamics. In contrast to the recycling route, transport to late endosomes is usually highly selective, accounting for the bulk of downregulated receptors but only a minor portion (?10%) of total internalized protein and lipid (Koval and Pagano, 1989; Trowbridge et al., 1993). Endosomes along these two pathways also exhibit marked ultrastructural differences. Whereas elements of the recycling pathway consist of very thin tubules (50C60 nm in diameter and up to several microns in length), endosomes at all stages of the degradation pathway exhibit a typical multivesicular appearance caused by the accumulation of internal membranes within their lumen, hence the name multivesicular body (MVB)1 (Dunn et al., 1986; Tooze and Hollinshead, 1991; Parton et al., 1992; van Deurs et al., 1993; Futter et al., 1996). Finally, these two pathways differ in their acidification properties. The lumenal pH decreases from 6.2 in early endosomes to 5.5 in endosomes of the degradation pathway but raises to 6.4 in recycling endosomes (Yamashiro et al., 1984; Mellman et al., 1986; Sipe and Murphy, 1987). It is intriguing how early endosomal membranes can give rise to elements that differ so widely in their business, internal milieu, and protein composition. In previous in vivo and in vitro studies, we have recognized and characterized intermediates (endosomal carrier vesicles [ECVs]), which mediate transport from early to late endosomes (Gruenberg et al., 1989; Bomsel et al., 1990; Aniento et al., 1993, 1996; Clague et al., 1994; Robinson et al., Mcl1-IN-9 1997). These vesicles exhibit a typical multivesicular ultrastructure and will be referred to as ECV/MVBs in this study. We found that the formation of Mcl1-IN-9 ECV/MVBs from early endosomes depends on coatomer protein (COP) (Aniento et al., 1996), a subunit of the COP-I coat previously shown to mediate anterograde and/or retrograde transport at early stages of the biosynthetic pathway (Orci et al., 1986; Ostermann et al., 1993; Pepperkok et al., 1993; Letourneur et al., 1994). Rabbit polyclonal to GPR143 Studies comparing endosomal and biosynthetic COPs, however, revealed that these exhibit, at least in part, different properties. Two subunits of the biosynthetic COP-I coat, and , are not present on endosomes (Whitney et al., 1995; Aniento et al., 1996), suggesting that the composition of the endosomal coat is simpler, or that this endosomal homologues of and have not been identified. In addition, we have observed that ECV/MVB formation from early endosomes depends on the acidic lumenal pH and that COP binding to early endosomes is usually itself pH dependent (Clague et al., 1994; Aniento et al., Mcl1-IN-9 1996). In contrast, COPs in the biosynthetic pathway are recruited onto the membranes of nonacidic organelles. Candidates responsible for COP-I binding to biosynthetic membranes include proteins normally retained in or retrieved to the endoplasmic reticulum via a KKXX motif (Cosson and Letourneur, 1994), as well as members of a novel family of biosynthetic membrane proteins in the 20C25-kD range (Fiedler et al., 1996; Sohn et al., 1996). Recent studies.