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

Electrophoresis
S MéresseJ P Gorvel

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 ...Continue Reading

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Citations

Aug 20, 2003·The Journal of Experimental Medicine·Jean CelliJean-Pierre Gorvel
Jul 29, 2010·PLoS Pathogens·Nina SchroederStéphane Méresse
Mar 12, 2005·Cellular Microbiology·Gilbert GreubStéphane Méresse
May 16, 2009·BMC Evolutionary Biology·Paweł Mackiewicz, Elzbieta Wyroba
Aug 6, 2004·Molecular Therapy : the Journal of the American Society of Gene Therapy·Christine GonçalvesChantal Pichon
Jan 10, 2002·The Journal of Biological Chemistry·Francois AuthierGilbert Briand
Nov 22, 2000·The Journal of Biological Chemistry·T YamazakiY Ihara

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