Mutational analysis of the Arf1*GTP/Arf GAP interface reveals an Arf1 mutant that selectively affects the Arf GAP ASAP1.
Arf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and alpha helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within alpha helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by approximately 10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, k(cat). In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs.
Luo
Ruibai
R
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Jacques
Kerry
K
Ahvazi
Bijan
B
Stauffer
Stacey
S
Premont
Richard T
RT
Randazzo
Paul A
PA
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics* / Q000378:metabolism*; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells
C470574:AGAP1 protein, human; C117891:ASAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors