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-COP
* Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002; and 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 Department of
Cell Biology, University of Geneva, CH-1211 Geneva 4, Switzerland
-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 -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 -COP cDNA demonstrating that they directly or indirectly reflected the -COP defect. Taken together, the
results suggest that -COP acts early in the endocytic
pathway, most likely inhibiting the normal sorting and
recycling functions of early endosomes.
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