Role of Coatomer and Phospholipids in GTPase-activating Protein-dependent Hydrolysis of GTP by ADP-ribosylation Factor-1

Abstract

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...

A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

“…This model is consistent with a result that Pap␤ exhibits efficient catalytic GAP activity for GTP-Arf1 in vitro (18). It should also be noted that there are several reports contradictory to Goldberg's model, suggesting that the coatomer may simply aid the productive association of Arf1 and ArfGAP1 (17,19,20). On the other hand, a study on the interface array of protein interactions of the small GTPases indicates that the protein-protein contacts in the Arf1-ArfGAP1 complex are significantly different from those found in other GAP and small GTPase complexes (21).…”

Systematic Structure-Function Analysis of the Small GTPase Arf1 in Yeast

“…Goldberg's model is based on the finding that the addition of coatomer to Arf1p and ArfGAP enhances GTP hydrolysis on Arf1p several orders of magnitude and it is suggested that coatomer may contribute an arginine finger residue. However, this model has been challenged by the finding that under certain conditions lipid can also accelerate the rate of GTP hydrolysis and that the addition of coatomer does not further increase this rate (Szafer et al, 2000). Also, the binding site for ArfGAP on Arf1p seems to be incompatible with the crystal structure of another ArfGAP, PAP␤.…”

“…Alternatively, they may indicate that residues implicated by crystallographic studies to interact with ArfGAP are not critical for the mechanism of GTP hydrolysis. Indeed, the precise mechanism by which ArfGAP catalyzes GTP hydrolysis on Arf proteins remains controversial (Goldberg, 1999;Mandiyan et al, 1999;Szafer et al, 2000). The structure of Arf1p complexed with ArfGAP was solved for the GDPbound form of Arf1p, the product of the reaction, so no direct information about the role of particular residues in the mechanism of GTP hydrolysis is available.…”

Golgi Localization Determinants in ArfGAP1 and in New Tissue-specific ArfGAP1 Isoforms

“…Proteins were expressed in the BL21/DE3 Escherichia coli strain harboring pLysS by induction for 3 h at 30°C in the presence of 0.1 mM isopropyl-␤-D-thiogalactopyranoside. Bacterial pellets were extracted with 6 M guanidine hydrochloride, and proteins were purified to near homogeneity by nickel-nitrilotriacetic acid chromatography followed by dialysis and Resource Q chromatography as previously described (27).…”

“…Whereas the catalytic domain of ArfGAP1 contains the machinery required for the catalysis of GTP hydrolysis (17,27), the non-catalytic part contains information for the targeting of ArfGAP1 to the Golgi, as indicated by the correct targeting of GFP fusions of non-catalytic fragments (28). The non-catalytic part mediates the interaction of ArfGAP1 with loosely packed lipids (24) and is also involved in protein-protein interactions, as demonstrated for the interaction of ArfGAP1 with the COPI cargo, the KDEL receptor (29,30).…”