Miriam Rotman

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.

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.

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.

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.

Requirement for both the amino-terminal catalytic domain and a noncatalytic domain for in vivo activity of ADP-ribosylation factor GTPase-activating protein.

The small GTP-binding protein ADP-ribosylation factor-1 (ARF1) regulates intracellular transport by modulating the interaction of coat proteins with the Golgi complex. Coat protein association with Golgi membranes requires activated, GTP-bound ARF1, whereas GTP hydrolysis catalyzed by an ARF1-directed GTPase-activating protein (GAP) deactivates ARF1 and results in coat protein dissociation. We have recently cloned a Golgi-associated ARF GAP. Overexpression of GAP was found to result in a phenotype that reflects ARF1 deactivation (Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305–7316). In this study, we used this phenotype to define domains in GAP that are required for its function in vivo. As expected, mutations in the amino-terminal part of GAP that were previously found to abolish ARF GAP catalytic activity in vitro abrogated ARF1 deactivation in vivo. Significantly, truncations at the carboxyl-terminal part of GAP that did not affect GAP catalytic activity in vitro also diminished ARF1 deactivation. Thus, a noncatalytic domain is required for GAP activity in vivo. This domain may be involved in the targeting of GAP to the Golgi membrane.

Golgi localization determinants in ArfGAP1 and in new tissue-specific ArfGAP1 isoforms.

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 reticulum-Golgi 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 in-frame 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.

33 - Expression, Purification, and Properties of ADP-Ribosylation Factor (ARF) GTPase Activating Protein-1

This chapter describes the expression, purification, and properties of adenosine diphopshate (ADP)-ribosylation factor (ARF) guanosine triphosphate (GTP)ase activating protein-1(GAP1). GAP1 is expressed at low level, comprising less than 0.01% of cytosolic proteins in all tissues examined so far. The protein can be obtained from rat liver cytosol. Because of the very low yield of pure protein and the current availability of methods for the purification of GAP1 from recombinant expression systems, the original purification procedure is outlined in the chapter. The entire catalytic domain of GAP1 can be expressed at high levels in E. coli and employed in biochemical and structural studies. The most consistent results are obtained in terms of expression level and GAP activity from constructs encoding the first 257 amino acids of GAP1. No expression is detected with constructs encoding more than the first 359 amino acids. Even though the catalytic part of GAP1 can be expressed in E. coli, expression of the noncatalytic carboxy-terminal part cannot be accomplished in this system. However, a full-length GAP1 protein can be prepared by using the baculovirus expression system.

Leishmania donovani: Characterization of a 38-kDa membrane protein that cross-reacts with the mammalian G-protein transducin

We investigated the presence in Leishmania donovani promastigotes of proteins with homology to the G-proteins known to mediate signal transduction in other organisms. [alpha 32P]GTP binding experiments revealed the presence in the promastigote membrane of GTP-binding sites with high affinity and specificity. Experiments with antisera directed against mammalian G-proteins showed that the promastigotes possess a 38-kDa protein (p38) which strongly reacts with an antiserum directed against a decapeptide containing the C-terminal sequence of transducin, the G-protein that mediates visual signal transduction. The interaction of p38 with the antiserum is specifically blocked by the decapeptide antigen. p38 is enriched in plasma membranes and is absent in cytosol and in a mitochondria-enriched fraction. p38 was also detected in two other Leishmania species, L. mexicana and L. major. The migration of p38 upon sucrose gradient centrifugation of detergent extract of L. donovani membranes corresponded to Mr of approximately 70,000, indicating that p38 is part of an oligomeric structure. The findings suggest that p38 may be a component of a transmembrane signal transduction system in Leishmania.

ARF GTPase-activating protein 1

Regulators of Arf activity include a family of proteins with a shared domain, the cysteine-rich Arf GAP domain, that is responsible for activating the latent GTPase activity of Arfs. The first of these to be discovered, Arf GAP1 is the focus of this chapter. It’s role in the cellular actions of Arfs, particularly vesicular traffic, and the regulation of Arf GAP1 by other factors, e.g., lipids, is discussed.

Preparation of 6-125I-labeled amiloride derivatives.

Amiloride and certain of its derivatives are effective inhibitors of Na/H antiporters and of epithelial Na channels. We describe a simple method for the preparation of a variety of pharmacologically active 6-iodoamiloride derivatives that are labeled with 125I at high specific radioactivity. 6-Dechloroamiloride derivatives (bearing a hydrogen atom instead of the chlorine at the 6 position of the amiloride molecule) are reacted with 125ICl, prepared by the oxidation of the iodide in Na125I preparations. The 125I-labeled derivatives are separated from free 125I by anion exchange chromatography, or purified by thin layer chromatography. Both 6-dechloroamiloride and 5-(N-alkyl)-6-dechloroamiloride derivatives can be labeled by this method, with yields varying between 10 and 70%, depending on the ICl concentration and the structure of the 5-N-alkyl group. Efficient radiolabeling at high specific radioactivity also depends on the use of freshly prepared batches of 125I. Using carrier-free 125I, [125I]6-iodoamiloride and [125I]6-iodo-5-(N-tert-butyl)amiloride were prepared with yields of 27 and 22%, respectively. Potential applications of the 125I-labeled amiloride derivatives include ligand binding and affinity labeling experiments.