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

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are func-tionally 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 condi-tional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associ-ated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive tem-perature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require deliv-ery 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 re-ceptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes re-distributed 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 re-quired 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 pheno-types were also corrected by transfection of wild-type