Hydrolysis of guanosine triphosphate (GTP) by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor-1 (ARF1) depends on a GTPase-activating protein (GAP). A complementary DNA encoding the ARF1 GAP was cloned from rat liver and predicts a protein with a zinc finger motif near the amino terminus. The GAP function required an intact zinc finger and additional amino-terminal residues. The ARF1 GAP was localized to the Golgi complex and was redistributed into a cytosolic pattern when cells were treated with brefeldin A, a drug that prevents ARF1-dependent association of coat proteins with the Golgi. Thus, the GAP is likely to be recruited to the Golgi by an ARF1-dependent mechanism.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes.

The small GTP-binding protein ARF plays an established role in the control of vesicular traffic and in the regulation of phospholipase D activity. Like other GTP binding proteins, ARF becomes activated upon the binding of GTP, whereas GTP hydrolysis acts as a turn-off signal. The fact that purified ARF proteins have negligible GTPase activity has suggested that GTP hydrolysis by ARFs is dependent on a GTPase-activating protein (GAP). Here we report the complete purification of an ARF GAP from rat liver cytosol. Advanced stages in the purification were carried out in the presence of denaturing agents, making use of an unusual conformational stability, or refolding capacity, of the GAP. The GAP was purified about 15,000-fold and was identified as a protein of 49 kDa. Partial amino acid sequence analysis showed that the GAP is a previously uncharacterized protein. Both crude and purified GAP migrated on a Superdex 200 column as a 200-kDa complex, suggesting a tetrameric structure. The purified ARF GAP was stimulated by phosphoinositides and was inhibited by phosphatidylcholine, similar to the results previously reported for a preparation from brain (Randazzo, P. A., and Kahn, R. A. (1994) J. Biol. Chem. 269, 10758). The availability of the ARF GAP molecule will advance the understanding of the regulation of the cellular processes in which ARF proteins participate.

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 Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulumGolgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204 -214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244 -2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue inframe insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.Membrane traffic in the endoplasmic reticulum-Golgi shuttle is mediated by the COPI 3 and COPII trafficking systems. The COPII system mediates the initial exit from the endoplasmic reticulum, whereas subsequent transport to the Golgi apparatus and retrograde Golgi to endoplasmic reticulum traffic involves COPI carriers. The COPI and COPII coats are composed of evolutionarily distinct sets of proteins yet follow similar pathways of coat assembly and disassembly (for recent reviews see Refs. 1-3). In both systems a small GTPase (Arf1 and Sar1 for COPI and COPII, respectively) plays a key regulatory role. Following activation by a guanine nucleotide exchange protein, the GTPase translocates from cytosol to the organelle membrane, where it initiates the process of coat formation by the direct binding of coat subunits (4 -6). The coat in turn recruits cargo and polymerizes causing membrane deformation to form a bud.In the second phase of the GTPase cycle of Arf1 and Sar1, bound GTP is hydrolyzed with the aid of a GTPase-activating protein (GAP). The hydrolysis of GTP is a prerequisite for coat dissociation, and its inhibition leads to the accumulation of coated vesicles that are prevented from fusing with the target membrane (4, 7). The GAP for Sar1 is a subunit of the first layer of the COPII coat, the Sec23/24 complex (8). ArfGAPs constitute a large family of proteins containing a hi...