GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.
ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.
Walker
M W
MW
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Bobak
D A
DA
Tsai
S C
SC
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D000227:Adenine Nucleotides / Q000494:pharmacology*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D008563:Membrane Lipids / Q000378:metabolism*; D008970:Molecular Weight; D016716:PC12 Cells; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding
D000227:Adenine Nucleotides; D002352:Carrier Proteins; D006150:Guanine Nucleotides; D008563:Membrane Lipids; D010743:Phospholipids; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors