An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes.
The Ras-related protein ADP-ribosylation factor 1 (ARF1) is a low molecular weight GTP binding protein, which in its GTP state supports the binding of coatomer, a cytosolic coat protein complex, to Golgi membranes. To create an "active" ARF, we constructed a point mutation in ARF1, Q71I, which was predicted to slow the rate of GTP hydrolysis. We demonstrate that Q71I, in contrast to wild type ARF1, exhibits a 2-3-fold increase in the half-life of ARF-GTP and is able to promote stable coatomer binding to Golgi membranes in the presence of GTP in vitro. Additionally, Q71I is able to support the binding of a significant amount of coatomer to membranes in the absence of added nucleotides, effectively bypassing the brefeldin A (BFA)-sensitive exchange activity. Furthermore, transfection of cells with Q71I, but not ARF1, renders the Golgi association of coatomer resistant to the effects of BFA in vivo. These observations provide compelling evidence that ARF1 is a necessary GTP binding protein that regulates the reversible binding of coat proteins to Golgi membranes and that the effects of BFA on this process in living cells must be a consequence of BFA's inhibition of guanine nucleotide exchange onto ARF1.
Teal
S B
SB
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Hsu
V W
VW
Peters
P J
PJ
Klausner
R D
RD
Donaldson
J G
JG
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D020126:Brefeldin A; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D020756:Coatomer Protein; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004351:Drug Resistance; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D020756:Coatomer Protein; D003517:Cyclopentanes; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors