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 dissocia tion of coatomer, requires GTP hydrolysis on ARF1, which depends on a GTPas e-activating protein (GAP). Recent studies demonstrate that when GAP activi ty is assayed in a membrane-free environment by employing an amino-terminal truncation mutant of ARF1 (Delta 17-ARF1) and a catalytic fragment of the ARF GTPase-activating protein GAP1, GTP hydrolysis is strongly stimulated b y coatomer (Goldberg, J., (1999) Cell 96, 893-902). In this study, we inves tigated 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 Delta 17-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 de pendent GAP, ASAP1. Using wild-type myristoylated ARF1 loaded with GTP in t he presence of phospholipid vesicles, GAP1 by itself stimulated GTP hydroly sis efficiently, and coatomer had no additional effect. Disruption of the p hospholipid vesicles with detergent resulted in reduced GAP1 activity that was stimulated by coatomer, a pattern that resembled Delta 17-ARF1 activity . Our findings suggest that in the biological membrane, the proximity betwe en ARF1 and its GAP, which results from mutual binding to membrane phosphol ipids, may be sufficient for stimulation of ARF1 GTPase activity.