Rab7 Regulates Late Endocytic Trafficking Downstream of Multivesicular Body Biogenesis and Cargo Sequestration

Phillip A Vanlandingham,Brian P Ceresa

doi:10.1074/jbc.m809277200

Abstract

The small molecular weight G-protein RAB7 is localized to both early and late endosomes and has been shown to be critical for trafficking through the endocytic pathway. The role of RAB7 in the endocytic pathway has been controversial, with some groups reporting that it regulates trafficking from early to late endosomes and others ascribing its role to trafficking between late endosomes and lysosomes. In this study, we use RNA interference to identify the exact step RAB7 regulates in the movement of the epidermal growth factor receptor (EGFR) from the cell surface to the lysosome. In the absence of RAB7, trafficking of the EGF.EGFR complex through the early endosome to the late endosome/multivesicular body (LE/MVB) does not change, but exiting from the LE/MVB is blocked. Ultrastructural analysis reveals that RAB7 is not required for formation of intraluminal vesicles of the LE/MVB, since RAB7-deficient cells have an increased number of enlarged LE/MVBs densely packed with intraluminal vesicles. Biochemical data indicate that the EGFR complex is sequestered in these intraluminal vesicles. Together, these data provide evidence that RAB7 is required for the transfer of cargo from the LE/MVB to the lysosome and for endocytic organelle maintenance.

Highlights

By following the postinternalization events of cell surface receptors, considerable work has been done to elucidate the molecular details of the endocytic pathway (2)
Following clathrin-mediated internalization, the endocytic pathway is composed of a series of dynamic stages that progressively shuttle cargo from clathrin-coated vesicles to early endosomes, to late endosomes/multivesicular bodies (LE/MVBs),2 and to lysosomes for degradation
We show that RAB7 is required for lysosomal degradation of the EGF1⁄7EGFR complex

Summary

Introduction

By following the postinternalization events of cell surface receptors, considerable work has been done to elucidate the molecular details of the endocytic pathway (2). Following clathrin-mediated internalization, the endocytic pathway is composed of a series of dynamic stages that progressively shuttle cargo from clathrin-coated vesicles to early endosomes, to late endosomes/multivesicular bodies (LE/MVBs), and to lysosomes for degradation. Each of these endocytic stages is defined by the morphology and protein composition of the organelle. Previous attempts to understand the function of RAB7 have relied primarily on overexpression of wild type or mutant RAB7 (11, 12, 15, 16) This approach carries the caveat that high levels of the exogenous protein increase the potential for nonphysiological interactions between an overexpressed RAB and downstream RAB effectors.

Methods

Findings

Conclusion