Role of coatomer and phospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-ribosylation factor-1.
The binding of the coat protein complex, coatomer, to the Golgi is mediated by the small GTPase ADP-ribosylation factor-1 (ARF1), whereas the dissociation of coatomer, requires GTP hydrolysis on ARF1, which depends on a GTPase-activating protein (GAP). Recent studies demonstrate that when GAP activity is assayed in a membrane-free environment by employing an amino-terminal truncation mutant of ARF1 (Delta17-ARF1) and a catalytic fragment of the ARF GTPase-activating protein GAP1, GTP hydrolysis is strongly stimulated by coatomer (Goldberg, J., (1999) Cell 96, 893-902). In this study, we investigated the role of coatomer in GTP hydrolysis on ARF1 both in solution and in a phospholipid environment. When GTP hydrolysis was assayed in solution using Delta17-ARF1, coatomer stimulated hydrolysis in the presence of the full-length GAP1 as well as with a Saccharomyces cerevisiae ARF GAP (Gcs1) but had no effect on hydrolysis in the presence of the phosphoinositide dependent GAP, ASAP1. Using wild-type myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles, GAP1 by itself stimulated GTP hydrolysis efficiently, and coatomer had no additional effect. Disruption of the phospholipid vesicles with detergent resulted in reduced GAP1 activity that was stimulated by coatomer, a pattern that resembled Delta17-ARF1 activity. Our findings suggest that in the biological membrane, the proximity between ARF1 and its GAP, which results from mutual binding to membrane phospholipids, may be sufficient for stimulation of ARF1 GTPase activity.
Szafer
E
E
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Pick
E
E
Rotman
M
M
Zuck
S
S
Huber
I
I
Cassel
D
D
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D020756:Coatomer Protein / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion
D002352:Carrier Proteins; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; D010743:Phospholipids; D019814:Myristic Acid; D006160:Guanosine Triphosphate; D001120:Arginine; D020823:ADP-Ribosylation Factor 1