Inhibition of Endosome Function in CHO Cells Bearing a Temperature-sensitive Defect in the Coatomer (COPI) Component e -COP

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit e -COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable e -COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type e -COP cDNA demonstrating that they directly or indirectly reflected the e -COP defect. Taken together, the results suggest that e -COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes. E ndocytosis is responsible for the internalization of extracellular fluid and macromolecules as well as plasma membrane proteins, receptors, and their bound ligands. Within early endosomes, this complex cargo must be sorted to ensure proper delivery to the appropriate intracellular destinations. Many receptors such as low density lipoprotein (LDL) 1 receptor and some ligands such as transferrin (Tfn) are recycled back to the plasma membrane, whereas bulk fluid and ligands that dissociate from their receptors because of acidic endosomal pH are targeted to lysosomes (Gruenberg and Maxfield, 1995; Mellman, 1996). Other sorting events lead from endosomes back to the Golgi complex, specialized storage compartments, or in polarized cells, a plasma membrane domain other than the domain of entry (Mellman, 1996). The mechanisms responsible for sorting in early endosomes remain largely unknown, but seem likely to involve the formation of transport vesicles or tubules that selectively include or exclude individual components. As found for sorting or transport events in other organelles, endosomal sorting may directly or indirectly involve the assembly of cytosolic coatomer proteins (COP). Several distinct coat complexes have been identified to be functionally important in molecular sorting and vesicle budding. These include clathrin and the clathrin adaptor proteins responsible for much of the transport of receptors from the plasma membrane and the trans -Golgi network to endosomes (Pearse and Robinson, 1990) and the COPI and COPII complexes, which are well known to play important roles in mediating transport through the secretory pathway. COPI was first identified as coating Golgi-derived transport vesicles (Orci et al., 1986, 1993) and shown to function in the transport between the ER and Golgi (Pepperkok et al., 1993). It is a multimeric complex termed E. Daro’s present address is Immunex Corporation, 51 University St., Se-