Interactions of rab5 with cytosolic proteins.
Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.
Kurzchalia
T V
TV
European Molecular Biology Laboratory, Heidelberg, Germany.
Gorvel
J P
JP
Dupree
P
P
Parton
R
R
Kellner
R
R
Houthaeve
T
T
Gruenberg
J
J
Simons
K
K
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000737:chemistry*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D010777:Photochemistry; D011506:Proteins / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins
D000199:Actins; D003432:Cross-Linking Reagents; D011506:Proteins; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins