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© The Rockefeller University Press, 0021-9525/1997//1183 $5.00
The Journal of Cell Biology, Volume 139, Number 5, , 1997 1183-1195

Article

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



Feng Gu*, Fernando Aniento*, Robert G. Parton{ddagger}, and Jean Gruenberg*

* Biochemistry Department, University of Geneva, 1211-Geneva-4, Switzerland; and {ddagger} 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 {varepsilon} coatomer protein ({varepsilon}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 {varepsilon}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 {varepsilon}COP, we find that a subcomplex of β, β', 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 GTP{gamma}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 {varepsilon}COP is missing. In contrast, all COP subunits, except obviously {varepsilon}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 β, β', and {zeta}COP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of {alpha} and/or {varepsilon}COP.

1. Abbreviations used in this paper: COP, coatomer protein; ECV, endosomal carrier vesicles; HB, homogenization buffer; LDL, low density lipoprotein; Man6P-R, mannose-6-phosphate receptor; MVB, multivesicular body; PNS, postnuclear supernatant; WT, wild-type.

We sincerely wish to thank Marie-Hélène Beuchat for her precious technical help. We wish to thank Monty Krieger for providing us with the ldlF cell line. We also wish to thank Ban-Hock Toh for the gift of anti-EEA1 antibodies, Bernard Hoflack for antibodies against the cation-independent mannose-6-phosphate receptor, Cordula Hater and Felix Wieland for the gift of antibodies against COPs, Jim Rothman for anticoatomer antibodies, Ari Helenius for anticalnexin antibodies, and Thomas Kreis for monoclonal antibodies against βCOP. We are also grateful to Thomas Harder and Volker Gerke for supplying us with the human transferrin receptor cDNA. We wish to thank Monty Krieger, Thomas Kreis, Gisou van der Goot, Katherine Bowers, and all members of the group for critically reading the manuscript and for helpful suggestions.

Address all correspondence to Jean Gruenberg, Biochemistry Department, 30 quai E. Ansermet, 1211-Geneva-4, Switzerland. Tel. and Fax (same number): +41-22-702.6464. E-mail: jean.gruenberg{at}biochem.unige.ch

F. Aniento's new address is Department Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Valencia. c/ Vicent Andrés Estellés, Burjassot (Valencia), Spain.


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