Flow cytometric sorting and biochemical characterization of the late endosomal rab7-containing compartment.
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.
Méresse
S
S
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, France.
André
P
P
Mishal
Z
Z
Barad
M
M
Brun
N
N
Desjardins
M
M
Gorvel
J P
JP
D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D018699:Coated Vesicles; D006224:Cricetinae; D011992:Endosomes / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D019204:GTP-Binding Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000032:analysis; D011498:Protein Precursors / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins
D010455:Peptides; D011498:Protein Precursors; D000090802:rab7 GTP-Binding Proteins; D017526:Receptor, IGF Type 1; D002402:Cathepsin D; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins