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J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/12/1183/13 $2.00
Volume 139, Number 5, December 1, 1997 1183-1195

Functional Dissection of COP-I Subunits in the Biogenesis of Multivesicular Endosomes

Feng Gu,* Fernando Aniento,* Robert G. Parton,Dagger and Jean Gruenberg*

* Biochemistry Department, University of Geneva, 1211-Geneva-4, Switzerland; and Dagger  Center for Microscopy and Microanalysis, Department of Physiology and Pharmacology, and Center for Molecular and Cellular Biology, University of Queensland, Queensland 4072, Brisbane, Australia

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in epsilon  coatomer protein (epsilon COP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although epsilon COP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of epsilon COP, we find that a subcomplex of beta , beta ', and zeta COP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPgamma S and sensitivity to the endosomal pH. Previous studies showed that gamma  and delta COP are not found on endosomes. However, alpha COP, which is normally present on endosomes, is no longer recruited when epsilon COP is missing. In contrast, all COP subunits, except obviously epsilon COP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas beta , beta ', and zeta COP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of alpha  and/or epsilon COP.


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