Comparative Activity of ADP-ribosylation Factor Family Members in the Early Steps of Coated Vesicle Formation on Rat Liver Golgi Membranes

doi:10.1074/jbc.272.7.4141

Comparative Activity of ADP-ribosylation Factor Family Members in the Early Steps of Coated Vesicle Formation on Rat Liver Golgi Membranes*

From the Department of Medicine, Washington University, St. Louis, Missouri 63110

^§ To whom correspondence should be addressed:
Dept. of Medicine, Washington University, 660 S. Euclid, Box 8125, St. Louis, MO 63110.
Tel.: 314-362-8801; Fax: 314-362-8826.

Abstract

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPγS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent K_m of 5 μM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

Footnotes

↵^‡ Recipient of a Howard Hughes Medical Institute Predoctoral Fellowship.
↵* This work was supported in part by National Institutes of Health Grant CA08759. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ARF

ADP-ribosylation factor

BFA

brefeldin A

Brij 58

20 cetyl ether

GEF

guanine nucleotide exchange factor

mARF

mammalian ADP-ribosylation factor

NMT

myristoyl-CoA:protein N-myristoyltransferase

PLD

phospholipase D

yARF

S. cerevisiae ADP-ribosylation factor

GTPγS

guanosine 5′-O-(thiotriphosphate)

DTT

dithiothreitol

BSA

bovine serum albumin

PAGE

polyacrylamide gel electrophoresis

COP

coatomer protein

TGN

trans-Golgi network.
- Received September 10, 1996.
- Revision received December 5, 1996.