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© The Rockefeller University Press, 0021-9525/1998//1075 $5.00
The Journal of Cell Biology, Volume 140, Number 5, , 1998 1075-1089

Article

Mutant Rab7 Causes the Accumulation of Cathepsin D and Cation-independent Mannose 6–Phosphate Receptor in an Early Endocytic Compartment



Barry Press*, Yan Feng*, Bernard Hoflack{ddagger}, and Angela Wandinger-Ness*

* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500; and {ddagger} Institut de Biolgie, Institut Pasteur de Lille, 59021 Lille, France

Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6–phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances ~50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB– mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.

Abbreviations used in this paper: AP, adaptor protein complex; CD-MPR, cation-dependent mannose 6–phosphate receptor; CI-MPR, cation-independent mannose 6–phosphate receptor; EEA1, early endosome antigen 1; lgp, lysosomal membrane glycoprotein; PNS, postnuclear supernatant.

We extend our thanks to the numerous individuals who kindly provided us with reagents and made this study possible. Drs. Manfred Gossen and Hermann Bujard generously provided the tetracycline-controlled gene expression system; Drs. Ban-Hok Toh, April Robbins, and Jean Gruenberg kindly supplied antisera. We are also indebted to Ms. Mary Slater Venkata for expert laboratory management throughout this project. We thank Drs. Stuart Kornfeld and Robert Lamb for valuable comments and discussions during the course of this study.

Address all correspondence to Angela Wandinger-Ness, Department of Biochemistry, Molecular Biology and Cell Biology, Hogan 2-100, 2153 North Campus Road, Northwestern University, Evanston, IL 60208-3500. Tel.: (847) 467-1173. Fax: (847) 491-2467. E-mail: w-ness{at}nwu.edu


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