Flow cytometric sorting and biochemical characterization of the late endosomal rab7-containing compartment

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Abstract
Rab7 is a small molecular weight GTPase that is known to be associated with late endocytic compartments. Studies in which wild‐type or mutant forms of this protein have been overexpressed in mammalian cells have indicated that rab7 plays a role in controlling membrane transport between late endocytic compartments. However, both the precise site(s) of action and localization of rab7 remain unclear. In the present study, we have used density‐gradient centrifugation in combination with a new epitope‐specific flow cytometric sorting method to isolate rab7‐containing vesicles from baby hamster kidney (BHK) cells. Electron‐micrographs of sorted elements showed a homogeneous population of vesicles that resembles late endosomes. The polypeptide composition of rab7‐containing vesicles was then analyzed by two‐dimensional (2‐D) gel electrophoresis. Rab7‐containing vesicles were enriched in the cation‐independent mannose 6‐phosphate receptor and especially in the precursor forms of cathepsin D. Taken together, these results show that the rab7‐containing vesicles are a component of the endocytic pathway that connects late endosomes and lysosomes and in which precursor forms of lysosomal hydrolases, segregated from their receptor, might be included.