Arf1 dissociates from the clathrin adaptor GGA prior to being inactivated by Arf GTPase-activating proteins.
The effectors of monomeric GTP-binding proteins can influence interactions with GTPase-activating proteins (GAPs) in two ways. In one case, effector and GAP binding to the GTP-binding protein is mutually exclusive. In another case, the GTP-binding protein bound to an effector is the substrate for the GTPase-activating protein. Here predictions for these two mechanisms were tested for the Arf1 effector GGA and ASAP family Arf GAPs. GGA inhibited Arf GAP activity of ASAP1, AGAP1, ARAP1, and Arf GAP1 and inhibited binding of Arf1.GTPgammaS to AGAP1 with K(i) values correlating with the K(d) for the GGA.Arf1 complex. ASAP1 blocked Arf1.GTPgammaS binding to GGA with a K(i) similar to the K(d) for the ASAP.Arf1.GTPgammaS complex. No interaction of GGA with ASAP1 was detected. Consistent with GGA sequestering Arf from GAPs, overexpression of GGA slowed the rate of Arf dissociation from the Golgi apparatus following treatment with brefeldin A. Mutational analysis revealed the amino-terminal alpha-helix and switch I of Arf1 contributed to interaction with both GGA and GAPs. These data exclude the mechanism previously documented for Arf GAP1/coatomer in which Arf1 is inactivated in a tripartite complex. Instead, termination of Arf1 signals mediated through GGA require that Arf1.GTP dissociates from GGA prior to interaction with GAP and consequent hydrolysis of GTP.
Jacques
Kerry M
KM
Laboratory of Cellular Oncology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nie
Zhongzhen
Z
Stauffer
Stacey
S
Hirsch
Dianne S
DS
Chen
Ling-Xin
LX
Stanley
Katherine T
KT
Randazzo
Paul A
PA
D016475:3T3 Cells; D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D020727:ADP-Ribosylation Factors / Q000737:chemistry* / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009154:Mutation; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; C407306:GGA adaptor proteins; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D005982:Glutathione Transferase; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors