ArfGAP1 is a 415 aa soluble protein that reversibly interacts with the Golgi apparatus (Cukierman et al. 1995). Originally isolated from rat liver cytosol (Makler et al. 1995), it is the founding member of the ArfGAP family of proteins, all characterized by a conserved catalytic domain containing a zinc finger model whose structure was solved by Goldberg (1999).
The substrate of ArfGAP1, Arf1, is a key regulator of the COPI system that mediates vesicular transport in the ER–Golgi shuttle. Upon GDP to GDP exchange, Arf1 associates with the Golgi membrane and recruits the heptameric COPI coat (coatomer), which in turn sorts cargo proteins and polymerizes to form the coat cage surrounding the vesicle. The subsequent hydrolysis of Arf-bound GTP is required for the release of coatomer from the membrane, a prerequisite for vesicle fusion. This reaction requires the action of a GAP. ArfGAP1 was the first Arf-directed GTPase-activating protein purified and cloned. Thirty one ArfGAP proteins are currently known in human, three of which – ArfGAP1, 2, and 3 – are thought to regulate COPI-mediated transport though the Golgi. Silencing of ArfGAP1 or a combination of ArfGAP2 and ArfGAP3 in HeLa cells does not decrease cell viability; however, silencing all three ArfGAPs causes cell death. In yeast, two ArfGAPs –GCS1, the orthologue of ArfGAP1, and Glo3, the orthologue of mammalian ArfGAP2/3 – have been implicated in COPI-mediated transport and were shown to function as an essential pair (Poon et al. 1999).
Function of ArfGAP1
Most published data implicate ArfGAP1 as a regulator of the COPI system. However, various and sometimes conflicting reports exist on the role of ArfGAP1 in the biogenesis and consumption COPI vesicles.
First indication for a role of ArfGAP1 in deactivation of Arf1 at the Golgi was provided by the finding that overexpression of ArfGAP1 in cells results in redistribution of the Golgi and its fusion with the ER (Huber et al. 1998), an effect that was known before to result from the deactivation of Arf1 by the drug brefeldin-A.
Reconstitution of COPI vesicles from Golgi membranes revealed that blocking GTP hydrolysis by the use of GTPγS (Serafini et al. 1991) or an activating mutant of Arf1 (Tanigawa et al. 1993) lead to the production of vesicles that cannot uncoat. These findings lead to the prediction that ArfGAP activity should trigger coatomer release from membranes. Subsequent studies, however, suggested that GTP hydrolysis on Arf1 is required for efficient uptake of cargo into vesicles (Lanoix et al. 1999; Nickel et al. 1998; Pepperkok et al. 2000), implying that GAP activity may also promote cargo sorting that occurs during vesicle formation. Lee et al. (2005) presented evidences for a role of ArfGAP1 in regulating the binding of coatomer to cargo proteins, indicating a direct role of ArfGAP1 in regulating cargo sorting. This study further demonstrated a requirement of ArfGAP1 catalytic activity for vesicle formation from Golgi membranes suggesting that ArfGAP1 plays a central role in coupling cargo sorting and vesicle formation. An additional function of ArfGAP1 in the vesicle fission was also proposed (Kartberg et al. 2010; Yang et al. 2006). Finally, ArfGAP1 has been shown to be involved in the regulation of asymmetric tethering between flat and curved membrane mediated by the Arf1 effector, GMAP210 (Drin et al. 2008).
Regulation of ArfGAP1 Activity
Experiments in vitro have suggested two mechanisms for the regulation of ArfGAP1 activity: stimulation by the COPI coat and by regulated recruitment to membranes.
The first mechanism concerning the role of coatomer was first described by Goldberg (1999). Using the catalytic domain of ArfGAP1 and a truncated Arf1 lacking the first 17 residues that can be loaded with GTP in the absence of lipids or detergents, Goldberg reported that the activity of the ArfGAP1 can be stimulated by up to 1,000-fold by coatomer. In contrast, investigating ArfGAP1 catalytic activity in liposomal system (Szafer et al. 2000) or on Golgi membranes (Szafer et al. 2001) using myristoylated membrane-bound Arf1 revealed only moderate or no stimulation by the addition of coatomer, respectively. Examination of the enzymology of ArfGAP1 suggested that coatomer allosterically regulates ArfGAP1 activity, affecting the affinity of ArfGAP1 for Arf-GTP but not the catalytic rate constant. These results further support the idea that coatomer has a regulatory role on the activity of ArfGAP1 (Luo and Randazzo 2008).
The second mechanism concerns the role of lipids. Although there is no evidence for direct interaction of ArfGAP1 with specific lipids, binding of ArfGAP1 to membranes results in increase in GAP activity by bringing it into proximity with its membrane-bound substrate, Arf1-GTP. The binding of ArfGAP1 to liposomes and its catalytic activity are both increased by chemical or physical conditions that create open spaces in the outer leaflet of the membrane bilayer such as the presence of diacylglycerols, phospholipids containing monounsaturated fatty acids (Antonny et al. 1997; Bigay et al. 2005), and high membrane curvature. Curvature-dependent activity of ArfGAP1 in vitro is of particular interest as it offers a mechanism that may be employed in vivo to ensure efficient targeting of ArfGAP1 to coated vesicles and/or to the highly curved rim of the Golgi cisternae where budding of COPI vesicles takes place. Bigay et al. (2005) have identified a motif in the center of ArfGAP1 that mediates the interaction with loosely packed lipids. This domain, termed ALPS (for ArfGAP1 lipid packing sensor) is unstructured in solution but in the presence of loosely packed lipids, hydrophobic residues in ALPS are inserted between lipid residues and ALPS folds into an amphipathic helix with serine/threonine residues forming the hydrophilic face. A function for ALPS–lipid interaction in vivo is suggested by the findings that the hydrophobic residues in ALPS are required for the interaction of ArfGAP1 with the Golgi apparatus (Parnis et al. 2006). In subsequent studies a second amphipathic motif – ALPS2 – with similar physicochemical characteristics was identified in ArfGAP1 (Levi et al. 2008; Mesmin et al. 2007). The two amphipathic motifs are separated by a short break and function cooperatively conferring liposome interaction and Golgi localization of ArfGAP1.
Mounting evidence implicates ArfGAP1 as critical regulator of the COPI system, yet its precise role has remained uncertain, with suggested functions ranging from an uncoating factor to an essential coat component required for cargo sorting and vesicle formation. While most studies have focused on the role of ArfGAP1 in the COPI system, ArfGAP1 also interacts with components of clathrin-coated carriers (including clathrin, AP-1, and AP-2), although the functional consequences of these interactions remains to be established.