We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24α2, β1, δ1, and γ3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24β1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

The binding of the coat protein complex, coatomer, to the Golgi is mediated by the small GTPase ADP-ribosylation factor-1 (ARF1), whereas the dissociation of coatomer, requires GTP hydrolysis on ARF1, which depends on a GTPase-activating protein (GAP). Recent studies demonstrate that when GAP activity is assayed in a membrane-free environment by employing an amino-terminal truncation mutant of ARF1 (⌬17-ARF1) and a catalytic fragment of the ARF GTPase-activating protein GAP1, GTP hydrolysis is strongly stimulated by coatomer (Goldberg, J., (1999) Cell 96, 893-902). In this study, we investigated the role of coatomer in GTP hydrolysis on ARF1 both in solution and in a phospholipid environment. When GTP hydrolysis was assayed in solution using ⌬17-ARF1, coatomer stimulated hydrolysis in the presence of the full-length GAP1 as well as with a Saccharomyces cerevisiae ARF GAP (Gcs1) but had no effect on hydrolysis in the presence of the phosphoinositide dependent GAP, ASAP1. Using wild-type myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles, GAP1 by itself stimulated GTP hydrolysis efficiently, and coatomer had no additional effect. Disruption of the phospholipid vesicles with detergent resulted in reduced GAP1 activity that was stimulated by coatomer, a pattern that resembled ⌬17-ARF1 activity. Our findings suggest that in the biological membrane, the proximity between ARF1 and its GAP, which results from mutual binding to membrane phospholipids, may be sufficient for stimulation of ARF1 GTPase activity. ARF1 GTPases play a key role in the regulation of vesicular trafficking of proteins among different compartments of the eukaryotic cell. In the early secretory system, the ARF1 protein regulates the interaction of the coatomer coat complex with Golgi membranes (1, 2). In the active GTP-bound form, ARF1 triggers the recruitment of coatomer (3-5) apparently by direct interaction with its ␤-and ␥-subunits (6). The subsequent dissociation of coatomer depends on GTP hydrolysis on ARF1 (7,8). The cycles of GTP binding and hydrolysis on ARF1 are controlled by two sets of cytosolic regulatory proteins. Activation of ARF1 is brought about by guanine nucleotide exchange proteins (9 -16) whereas GTP hydrolysis depends on GTPaseactivating proteins (GAPs). ARF GAPs are a family of proteins sharing a catalytic domain of 120 -140 amino acids that includes a Cys 4 -zinc finger motif. The first ARF GAP to be discovered (GAP1) is a 45-kDa protein that distributes between the cytosol and Golgi complex and functions in the regulation of membrane traffic through this organelle (17-19). Saccharomyces cerevisiae contains two proteins (Gcs1 and Glo3) that show high similarity to GAP1 and possess ARF GAP activity (20, 21). The two yeast GAPs form an essential pair with a redundant function in the endoplasmic reticulum-Golgi shuttle. Recently, additional ARF GAPs belonging to two subfamilies were identified in mammalian cells. GIT1 is a 95-kDa protein from rat that interacts with GRK2 and regulates ␤ 2 -adrenergic re...

The interaction of the coatomer coat complex with the Golgi membrane is initiated by the active, GTP-bound state of the small GTPase ADP-ribosylation factor 1 (ARF1), whereas GTP hydrolysis triggers coatomer dissociation. The hydrolysis of GTP on ARF1 depends on the action of members of a family of ARF1-directed GTPase-activating proteins (GAPs). Previous studies in well defined systems indicated that the activity of a mammalian Golgi membrane-localized ARF GAP (GAP1) might be subjected to regulation by membrane lipids as well as by the coatomer complex. Coatomer was found to strongly stimulate GAP-dependent GTP hydrolysis on a membrane-independent mutant of ARF1, whereas we reported that GTP hydrolysis on wild type, myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles was coatomer-independent. To investigate the regulation of ARF1 GAPs under more physiological conditions, we studied GTP hydrolysis on Golgi membrane-associated ARF1. The activities at the Golgi of recombinant GAP1 as well as coatomer-depleted fractions from rat brain cytosol resembled those observed in the presence of liposomes; however, unlike in liposomes, GAP activities on Golgi membranes were approximately doubled upon addition of coatomer. By contrast, endogenous GAP activity in Golgi membrane preparations was unaffected by coatomer. Cytosolic GAP activity was partially reduced following immunodepletion of GAP1, indicating that GAP1 plays a significant although not exclusive role in the regulation of GTP hydrolysis at the Golgi. Unlike the activities of the mammalian proteins, the Saccharomyces cerevisiae Glo3 ARF GAP displayed activity at the Golgi that was highly dependent on coatomer. We conclude that ARF GAPs in themselves can efficiently stimulate GTP hydrolysis on ARF1 at the Golgi, and that coatomer may play an auxiliary role in this reaction, which would lead to an increased cycling rate of ARF1 in COPI-coated regions of the Golgi membrane.The budding of vesicles mediating the transport of proteins among different compartments of the secretory system is driven by the attachment of specialized protein complexes termed coats to the cytoplasmic surface of the donor membrane. These coat complexes also function in the sorting of appropriate cargo into the transport vesicle (reviewed in Refs.