Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro.
Galpha-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for Galpha subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Galphai proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5x) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Galphai3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Galphai3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Galphai3 and that membrane-associated GAIP is capable of interacting with Galphai3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.
Fischer
T
T
Division of Cellular and Molecular Medicine and Department of Pathology, University of California, San Diego, La Jolla, CA 92093-0651, USA.
Elenko
E
E
McCaffery
J M
JM
DeVries
L
L
Farquhar
M G
MG
D000818:Animals; D001692:Biological Transport; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D020710:RGS Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
D002966:Clathrin; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; D020710:RGS Proteins; D011994:Recombinant Proteins; C097187:regulator of G-protein signalling 19; D020558:GTP Phosphohydrolases