Dan Cassel

The ARF1 GTPase-Activating Protein: Zinc Finger Motif and Golgi Complex Localization

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.

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles a role for ArfGAP1

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 COPI system: Molecular mechanisms and function

Transport of membranes and proteins in eukaryotic cells is mediated by vesicular carriers. Here we review the biogenesis and functions of COPI vesicles, carriers that operate in the early secretory pathway. We focus on mechanisms mediating coat recruitment, uptake of cargo, vesicle budding and fission, and finally dissociation of the coat. In this context, recent findings on the interplay between machinery and auxiliary proteins in COPI vesicle formation and function will be discussed. Specifically, we will weigh the pros and cons of recent data on roles of the small GTP binding protein Arf1, of Arf1GAPs, and lipids during COPI carrier formation.

The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPase-activating protein for ARF1.

The small GTPase ADP‐ribosylation factor 1 (ARF1) is a key regulator of intracellular membrane traffic. Regulators of ARF1, its GTPase‐activating protein (GAP) and its guanine nucleotide exchange factor have been identified recently. However, it remains uncertain whether these regulators drive the GTPase cycle of ARF1 autonomously or whether their activities can be regulated by other proteins. Here, we demonstrate that the intracellular KDEL receptor, ERD2, self‐oligomerizes and interacts with ARF1 GAP, and thereby regulates the recruitment of cytosolic ARF1 GAP to membranes. Because ERD2 overexpression enhances the recruitment of GAP to membranes and results in a phenotype that reflects ARF1 inactivation, our findings suggest that ERD2 regulates ARF1 GAP, and thus regulates ARF1‐mediated transport.

Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase‐activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)–Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Δ and gcs1Δ single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER–Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v‐SNARE family that functions within the ER–Golgi alleviates the effects of a glo3Δ mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER.

Activation of ADP-ribosylation Factor 1 GTPase-Activating Protein by Phosphatidylcholine-derived Diacylglycerols

Disassembly of the coatomer from Golgi vesicles requires that the small GTP-binding protein ADP-ribosylation factor 1 (ARF1) hydrolyzes its bound GTP by the action of a GTPase-activating protein. In vitro, the binding of the ARF1 GTPase-activating protein to lipid vesicles and its activity on membrane-bound ARF1GTP are increased by diacylglycerols with monounsaturated acyl chains, such as those arising in vivo as secondary products from the hydrolysis of phosphatidylcholine by ARF-activated phospholipase D. Thus, the phospholipase D pathway may provide a feedback mechanism that promotes GTP hydrolysis on ARF1 and the consequent uncoating of vesicles.

Consensus nomenclature for the human ArfGAP domain-containing proteins

At the FASEB summer research conference on “Arf Family GTPases”, held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

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

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 (Δ17-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 Δ17-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 Δ17-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.

Spatiotemporal dynamics of the COPI vesicle machinery

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase‐activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence‐correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D ≈ 15 μm2 s−1), whereas coatomer diffuses 5–10 times more slowly than expected (D ≈ 1 μm2 s−1). This slow diffusion causes diffusion‐limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.

Regulation of GTP Hydrolysis on ADP-ribosylation Factor-1 at the Golgi Membrane

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.