Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component epsilon-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 epsilon-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 epsilon-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 epsilon-COP cDNA demonstrating that they directly or indirectly reflected the epsilon-COP defect. Taken together, the results suggest that epsilon-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.
Daro
E
E
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA.
Sheff
D
D
Gomez
M
M
Kreis
T
T
Mellman
I
I
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D020756:Coatomer Protein; D006224:Cricetinae; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D012672:Semliki forest virus / Q000378:metabolism; D013696:Temperature; D014168:Transferrin / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism
D015703:Antigens, CD; D020756:Coatomer Protein; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014168:Transferrin