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

Jennifer Ostrom Liang,Stuart Kornfeld

doi:10.1074/jbc.272.7.4141

Jennifer Ostrom Liang, Stuart Kornfeld

Open Access

https://doi.org/10.1074/jbc.272.7.4141

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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 GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM. 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.

Highlights

The family of mammalian ADP-ribosylation factors (ARFs) has been grouped into three classes based on amino acid sequence [9]
The activated, membrane-bound ARF in turn facilitates the recruitment of the AP-1 adaptor complex and the COPI coat complex onto the Golgi membranes
By comparing the ability of the Golgi membrane guanine nucleotide exchange factor (GEF) to activate mammalian ARFs (mARFs) 1, 5, and 6, we have determined that it has specificity for mARF1

Summary

EXPERIMENTAL PROCEDURES

Materials—Brefeldin A (BFA), protease inhibitors, trypsin, DTT, antibiotics, 20 cetyl ether (Brij 58), ATP, and GDP were obtained from Sigma; GTP and GTP␥S from Boehringer Mannheim; [35S]GTP␥S was from DuPont NEN; [␣-32P]GTP and [3H]myristic acid were from ICN Biomedicals Inc (Irvine, CA); Superdex-75 prep grade, DEAE-Sepharose Fast Flow, SP-Sepharose Fast Flow, and the SDS-PAGE low molecular weight markers were purchased from Pharmacia Biotech Inc.; nitrocellulose was from Schleicher & Schuell; enhanced chemiluminescence reagents for chemiluminescence were purchase from Amersham Corp.; isopropyl-1-thio-␤-D-galactopyranoside was from Amresco (Solon, OH); myristic acid was from Nu Chek Prep (Elysian, MN). The bovine ARF1 expression vector pOW12 [14] was a gift from Richard Kahn (Emory University, Atlanta). PET vectors (Stratagene, La Jolla, CA) containing the cDNAs for human ARFs 5 and 6 [15] were a gift from Richard Klausner (National Institutes of Health, Bethesda).

Comparative Activity of ARFs in Golgi Coated Vesicle Formation

RESULTS

Golgistimulated exchange

DISCUSSION