Snapshots of ARF1: Implications for Mechanisms of Activation and Inactivation

The minimum domain of rat p47 ARF GAP containing GAP activity for ARF is approximately 130 N-terminal amino acids that contain the zinc finger motif that is a signature for ARF GAP proteins (Cukierman et al. 1995xCukierman, E, Huber, I, Rotman, M, and Cassel, D. Science. 1995; 270: 1999–2002Crossref | PubMedSee all ReferencesCukierman et al. 1995). In a recent article in Cell, Goldberg 1999xGoldberg, J. Cell. 1999; 96: 893–902Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesGoldberg 1999 has provided us with the crystal structure of ARF-GDP bound to the 130–amino acid GAP catalytic domain. This structure represents the product complex of the GTPase reaction and so only inferences can be drawn about the active site alignment in the transition state where hydrolysis would occur. The structure is remarkable and unlike the complexes between other GAP proteins and G proteins large or small. For other Ras-related proteins, the GAP-binding domain overlaps the effector-binding site, precluding simultaneous interactions between the GTP-binding protein, effector, and GAP. In those proteins, the hydrolysis reaction is accelerated by residues from the GAP protein, usually including an arginine, that extend into the nucleotide-binding pocket of the G protein (24xTesmer, J.J.G, Berman, D.M, Gilman, A.G, and Sprang, S.R. Cell. 1997; 89: 251–261Abstract | Full Text | Full Text PDF | PubMedSee all References, 16xScheffzek, K, Ahmadian, M.R, and Wittinghofer, A. Trends Biochem. Sci. 1998; 23: 257–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (302)See all References). In contrast, the GAP-binding site on ARF does not overlap with its effector-binding site, nor does ARF GAP supply any residues to the active site that could be involved in hydrolysis (Figure 2Figure 2). The major direct contribution of ARF GAP to the hydrolysis reaction appears to be through stabilizing the switch 2 region of ARF to align glutamine 71 to participate in the hydrolysis reaction. With all of these differences from the standard mechanism by which GAP proteins act on small G proteins, how then does ARF GAP accelerate GTP hydrolysis?Figure 2Structure of the ARF1–ARF GAP Complex(A) Ribbon diagram of human ARF1 bound to the catalytic domain of rat ARF-GAP.(B) The complex is rotated 90° about the vertical axis.(Reprinted from Goldberg 1999xGoldberg, J. Cell. 1999; 96: 893–902Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesGoldberg 1999.)View Large Image | View Hi-Res Image | Download PowerPoint SlideWhen coatomer is added to ARF-GTP and the GAP, the GTPase reaction in vitro is accelerated 1000-fold (Goldberg 1999xGoldberg, J. Cell. 1999; 96: 893–902Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesGoldberg 1999). One simple mechanism for this rate enhancement is for coatomer to provide the residues that enter the active site and accelerate hydrolysis. It is possible that it is the complex of coatomer and ARF that is recognized by the GAP, leading to an orderly sequence of events compatible with prevailing ideas of the role of ARF GAP. However, if the interaction of GAP with ARF leads to a slow rate of hydrolysis, there is no reason a priori to propose that the GAP interaction with ARF must follow the coatomer interaction. Another possibility is that the GAP interacts with ARF earlier than coatomer, and that the complex of ARF and GAP provides the binding site for the coat proteins (see also Zhu et al. 1998xZhu, Y.X, Traub, L.M, and Kornfeld, S. Mol. Biol. Cell. 1998; 9: 1323–1337Crossref | PubMedSee all ReferencesZhu et al. 1998 and Springer et al. 1999xSpringer, S, Spang, S, and Schekman, R. Cell. 1999; 97: 145–148Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesSpringer et al. 1999). Once the coat has formed and become stable (through interlocking subunits perhaps) ARF might hydrolyze GTP and both ARF and GAP might leave the coat, possibly to recycle for more rounds of coatomer addition.Rather than merely confirming what we thought that we knew, the snapshots of ARF provided to us by Goldberg challenge our ideas of how ARF works. Clearly structures of additional complexes in the pathway of ARF action will provide major insight into its mechanism. One of these will certainly be the trimeric complex of ARF, ARF GAP, and coatomer subunits. Others will require that the major technical hurdle of the insolubility of myristoylated full-length ARF is solved, so that, among other things, we might learn how nucleotide hydrolysis on ARF can be coupled to extraction of the amino terminus of ARF from the membrane. The uniqueness of the ARF exchange and GAP reactions in relation to those that have been studied with other G proteins reflects the fact that the membrane is also a player in the cycle of ARF activation and inactivation (Goldberg 1998xGoldberg, J. Cell. 1998; 95: 237–248Abstract | Full Text | Full Text PDF | PubMed | Scopus (356)See all ReferencesGoldberg 1998). Why this is the case, and why ARF is myristoylated whereas Sar1p, a small G protein with a similar function, is not, remain major questions for the future.*e-mail: michael.roth@email.swmed.edu.