Effects of brefeldin A and accessory proteins on association of ADP-ribosylation factors 1, 3, and 5 with Golgi.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. By cDNA and genomic cloning, at least six mammalian ARFs were identified. Brefeldin A (BFA) disrupts Golgi membranes and inhibits binding of soluble high molecular weight proteins to Golgi fractions. We examined the effects of BFA on binding of ARF1, -3, and -5 to a Golgi fraction in the presence of an ATP-regenerating system and a fraction of soluble, high molecular weight, accessory proteins (SAP), presumably containing complexes identified by others as coatomers that are involved in vesicular transport. ARF binding in all instances was dependent on guanosine 5'-O-(3-thiotriphosphate) and increased by the ATP-regenerating system. Binding of ARF1 and -3, but not ARF5, was enhanced by SAP. BFA inhibited the SAP-dependent, but not the SAP-independent, binding of ARF1 and -3. It had no effect on the increment in binding produced by an ATP-regenerating system. B36, an inactive derivative of BFA, did not inhibit SAP-dependent binding of ARF1 and -3. Binding of ARF5, which was SAP-independent, was not affected by BFA. These observations are consistent with the conclusion that mammalian ARFs differ in their dependence on accessory proteins for interaction with Golgi and, perhaps, other cellular membranes and that BFA specifically inhibits SAP-dependent ARF binding.
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
S C
SC
Tsai
R
R
Adamik
R S
RS
Haun
J
J
Moss
M
M
Vaughan
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D001921:Brain / Q000378:metabolism*; D020126:Brefeldin A; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003517:Cyclopentanes / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats
D002352:Carrier Proteins; D003517:Cyclopentanes; D009419:Nerve Tissue Proteins; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C486478:Arf5 protein, rat