REQUIREMENT FOR BOTH THE AMINO-TERMINAL CATALYTIC DOMAIN AND A NONCATALYTIC DOMAIN FOR IN-VIVO ACTIVITY OF ADP-RIBOSYLATION FACTOR GTPASE-ACTIVATING PROTEIN

The small GTP-binding protein ADP-ribosylation factor-1 (ARF1) regulat es intracellular transport by modulating the interaction of coat prote ins with the Gels complex, Coat protein association with Gels membrane s requires activated, GTP-bound ARF1, whereas GTP hydrolysis catalyzed by an ARF1-directed GTPase-activating protein (GAP) deactivates ARF1 and results in coat protein dissociation. We have recently cloned a Go lgi-associated ARF GAP. Overexpression of GAP was found to result in a phenotype that reflects ARF1 deactivation (Aoe, T,, Cukierman, E,, Le e, A., Cassel, D,, Peters, P, J,, and Hsu, V, W, (1997) EMBO J. 16, 73 05-7316), In this study, we used this phenotype to define domains in G AP that are required for its function in vivo, As expected, mutations in the amino-terminal part of GAP that were previously found to abolis h ARF GAP catalytic activity in vitro abrogated ARF1 deactivation in v ivo. Significantly, truncations at the carboxyl-terminal part of GAP t hat did not affect GAP catalytic activity in vitro also diminished ARF 1 deactivation. Thus, a noncatalytic domain is required for GAP activi ty in vivo. This domain may be involved in the targeting of GAP to the Golgi membrane.