by Richard A. Kahn, Laboratory of Biological Chemistry, NCI


Molecules important in Arf signaling: a heterotrimeric protein, Gs; Arf GAP; phospholipase D (PLD); and a heptameric complex, coatomer. Translocation of the Arf and coatomer from cytosol to membrane-bound forms is indicated by the arrows. Interactions of Arf, Arf GAP, and PLD with specific phospholipids are also indicated within the boxed area, which represents a membrane bilayer. (PA, phosphatidic acid; PC, phosphatidylcholine; PIP2, phosphatidylinositol-4,5-bisphosphate)

Most recent reviews of the properties of biological membranes convey one of two overly narrow views. Some portray biological membranes as molecular putty readily fashioned into different shapes (vesicles, tubes, and sheets) by proteins or protein complexes. Others portray the biophysical properties of the amphipathic lipids themselves as sufficient to explain all the diverse and dynamic changes occurring at each biological membrane surface. A remarkable convergence of recent information from cell biology, neurobiology, and studies of the yeast secretory pathway have yielded several proteins (e.g., NSF, SNAPs, and synaptobrevins) that appear to serve a common role in membrane traffic, synaptic transmission, and organelle remodeling (1). Although this exciting development should not be minimized, what is lacking from the emerging models of regulated and constitutive endo- and exocytosis is an integrated view encompassing the roles of these proteins and of the lipid components in membranes.

One reason this synthesis has not emerged is that we simply don't know how the cell "reads" and processes information inherent in the lipid composition of a membrane and integrates it into interactions with integral and peripheral membrane proteins. Now, a family of regulatory GTP-binding proteins has been observed to possess many of the features predicted for integrators of such interactions. These proteins, called ADP-ribosylation factors, or Arfs, have recently been found to 1) interact with specific lipid components of membranes, 2) modify the lipid composition and release potential second messengers through activation of phospholipase D (PLD), and 3) regulate the assembly of at least a subset of protein complexes or membrane coats (for reviews, see refs. 2 and 3). In short, Arf proteins appear to be both sensors of the lipid environment and transducers of information (including information inherent in lipids), which results in changes in Arf activities and the consequent assembly of protein structures on the membranes.

Genes for GTP-binding Proteins
The name ADP-ribosylation factor derives from the discovery, in 1984, of the factor that confers sensitivity of the stimulator of adenylate cyclase (Gs) to cholera toxin, an ADP-ribosyltransferase (4). In the ensuing years, the number of distinct genes discovered that encode low-molecular-weight GTP-binding proteins has increased to more than 100. The products of these genes can be conveniently (but not always accurately) lumped into four sub-families of the Ras superfamily: the Ras, Rab, Rho, and Arf proteins. Six mammalian Arf proteins have been identified, five of which appear to be expressed in all cells and tissues; Arf2 has, so far, been found only in cows. Arfs are ubiquitous in eukaryotes, are very highly conserved both structurally and functionally, and exchange guanine nucleotides in a complex, highly regulated fashion that is sensitive to lipids, salts, and divalent metals. A larger sub-group in the Arf family is the Arf-like proteins, which are structurally related (typically 40-60% identical to any Arf) but lack defined Arf activities, such as acting as the cholera toxin cofactor for Gs (5).

A role for Arf proteins in membrane traffic has now been confirmed by several independent techniques. Arf proteins' role in protein secretion was first observed in the yeast Saccharomyces cerevisiae (6). Localization of Arf proteins to the cytosolic side of (predominantly) cis-Golgi structures, and later other Golgi-related structures, further supports the conclusion that a major site of Arf action in mammalian cells is the Golgi complex. However, Arf plays other roles in membrane traffic, and its other sites of action now include the endoplasmic reticulum, the nuclear envelope, and elements of the endocytic pathway, likely the plasma membrane and early endosomes. We learned this from in vitro assays of membrane traffic, including those for intra-Golgi transport and nuclear vesicle and endosome fusion, which are inhibited by GTPgS and require the addition of cytosol or a cytosolic factor (7,8). In two of these cases (intra-Golgi transport and nuclear vesicle fusion), the cytosolic factor was purified to homogeneity and shown unambiguously to be an Arf protein. Independent studies have confirmed roles for Arf proteins in the movement of proteins out of the ER and Golgi and in endocytosis. Expression of dominant activating alleles of Arf1 blocks proteins from exiting the ER (9) and causes dramatic expansion of the ER lumen (10), expansion and vesiculation of the Golgi stacks with inhibition of protein secretion (10), and loss of fluid-phase endocytosis (10). These data indicate that Arf proteins have dynamic roles in the maintenance of the integrity of several organelles, particularly the Golgi and ER.

Arfs' Role in Membrane Traffic
Although more sites of Arf action have been discovered over the past 10 years, details of the molecular actions of Arf proteins remain poorly understood. Some insights have come from the recognition of the roles of coat proteins in membrane traffic in general and in Arf action in particular. Arf proteins have been found on both clathrin-coated (11) and non-clathrin-coated, or Golgi-derived (12), vesicles. The major constituent of nonclathrin-coated vesicles is a protein complex, termed coatomer. Coatomer has been purified from cytosol and is made up of seven coat proteins (designated a through z COP), which appear to exist as a stable heptamer, cycling between soluble and particulate pools (13). A role for Arf proteins as regulators of the reversible binding of coatomer to Golgi-enriched membranes is implied by the demonstration that when cytosol is depleted of Arf proteins it fails to support coatomer binding, whereas addition of purified recombinant Arf1 is sufficient to restore GTPgS-stimulated coatomer association with membranes (14). In addition to the observation that in vitro binding of coatomer to membranes appears to require Arf, the Arf itself has been shown to bind reversibly to phospholipid micelles and membranes in a GTP-dependent manner (15). Thus, regulated binding of guanine nucleotides to Arf is proposed to control the association of coat proteins with membranes.

Arf proteins have three biochemical properties that distinguish them from all other GTP-binding proteins: 1) GTP binding is highly dependent on phospholipids (16), 2) the activated protein, Arf*GTP, stably associates with phospholipid micelles or membranes whereas Arf*GDP is soluble (15), and 3), in the absence of GTPase activiting Protein (GAP), purified Arf proteins have no detectable intrinsic GTPase activity (17). These observations support the idea that the reversible interaction with membrane phospholipids and the binding of guanine nucleotides are highly integrated.

Our understanding of Arf action changed dramatically last year with the demonstration that Arf is an activator of phospholipase D (PLD), the membrane-bound enzyme that catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline (18,19). PLD has been implicated as an effector of several growth factors, though the roles of phospholipases C and D are somewhat confused as a result of the cell's ability to interconvert the products of the two lipases (20). Brown et al. (18) found that phosphatidylinositol-4,5-bisphosphate (PIP2) is required for Arf stimulation of PLD activity and that Arf-dependence of PLD activity was maintained through several steps of purification, suggesting a direct interaction between Arf and PLD. The activation of a specific enzymatic activity by Arf provides the first enzymatic effector for Arf actions and contrasts with the view of Arf as a structural protein constituent of organelle or vesicle coats.

Further evidence of a complicated interrelationship between Arf action and membrane phospholipids is provided by research on Arf GAP. GAP activates the latent GTPase activity, which leads to the termination of the activated state of Arf. For at least one other GTP-binding protein, Gq-stimulated PLC-b, the effector was also shown to act as a GAP (21). The initial characterization of a specific Arf GAP was recently reported by Randazzo and Kahn (17). Arf GAP activity is highly dependent on PIP2, can be further stimulated by PA, the product of PLD, and is inhibited by PC, the substrate of PLD.

The requirement for PIP2 in two different Arf-dependent assays -- for PLD and Arf GAP -- suggested a potential common site of action for this acid phospholipid. Direct effects of PIP2 on Arf proteins are described in Terui et al. (22) and include an increase greater than 100-fold in the rate of GDP dissociation and, paradoxically, loss of GTP binding. PIP2 promotes the dissociation of GDP and binds to and stabilizes the nucleotide-free form of the protein. These effects of PIP2 on Arf are homologous to the actions of the guanine nucleotide exchange factor, Cdc25, on Ras (23). The specificity and magnitude of the effects of phospholipids on Arf properties in vitro make it very likely that these same interactions are important in vivo and raise the possibility that PIP2 may play the role of a nucleotide-exchange factor for Arf in cells.

Structural and mutagenesis studies have begun to reveal details of several specific Arf interactions. Arf has at least two distinct protein-binding domains, one that re-quires an intact amino terminus that is required for Gs and Arf GAP binding and one that is less sensitive to deletion of the amino terminus and to which cholera toxin binds (24). In addition, Arf has two distinct phospholipid-binding sites. One is a relatively non-specific site that requires an intact amino terminus, confers lipid dependence on nucleotide binding, and helps to stabilize the active form of the protein. The other is a highly specific PIP2 site whose occupancy promotes stability of the apoprotein (22). Comparisons of the crystal structures of Ras and Arf are now possible because the Arf*GDP structure has been solved (25). This model will allow directed studies of the residues involved in Arf's interactions and will provide a structural framework on which to build further models to explain all of Arf's activities.

The physiological relevance of our observations on Arf remain to be established, but it seems reasonable to suggest that several of the key regulators and effectors of Arf actions are those identified by the in vitro studies. When the actions of any Arf protein are being consided, the proximate membrane and its specific lipid composition must now also be considered. Specific interactions between a single protein and lipid components of biological membranes as well as cellular proteins are not limited to members of the Arf family. By combining structural studies with the many functional assays of Arf activity now available, we hope to be able to describe in detail a molecular mechanism that may serve as a model for other proteins that interact reversibly with membranes. Such information may ultimately prove valuable in studies of the etiology and potential repair of defects in cellular physiology associated with a variety of human diseases.

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2. R.A. Kahn. "The Biology of ADP-ribosylation Factors." In: Handbook of Experimental Pharmacology: GTPases in Biology, B. Dickey and L. Birnbaumer, eds. Heidelberg: Springer-Verlag, pp. 529 - 42 (1993).

3. J. Moss and M. Vaughan. "ADP-ribosylation factors, 20,000 M(r) guanine nucleotide - binding protein activators of cholera toxin and components of intracellular vesicular transport systems."Cell Signaling 5, 367 - 69 (1993).

4. R.A. Kahn and A.G. Gilman. "Effects of cholera toxin-dependent ADP- ribosylation on the regulatory component of adenylate cyclase." J. Biol. Chem. 259, 6235 - 40 (1984).

5. J. Clark, L. Moore, A. Krasinskas, J. Way, J. Battey, J. Tamkun, et al. "Selective amplification of novel members of the ADP-ribosylation factor (ARF) family: cloning of new human and Drosophila ARL genes." Proc. Natl. Acad. Sci. USA 90, 8952 - 56 (1993).

6. T. Stearns, M.C. Willingham, D. Botstein, and R.A. Kahn. "The ADP-ribosylation factor is functionally and physically associated with the Golgi complex." Proc. Natl. Acad. Sci. USA 87, 1238 - 42 (1990).

7. P. Melancon, B.S. Glick, V. Malhotra, P.J. Weidman, T. Serafina, M.L. Gleason, et. al. "Involvement of GTP-binding `G' Proteins in transport through the Golgi stack." Cell 51, 1053 - 62 (1987).

8. A.L. Boman, M.R. Delannoy, and K.L. Wilson. "GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro." J. Cell Biol. 116, 281 - 94 (1992).

9. C. Dascher and W.E. Balch. "Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus." J. Biol. Chem. 269, 1437 - 48 (1994).

10. C.J. Zhang, A.G. Rosenwald, M.C. Willingham, S. Skuntz, J. Clark, and R.A. Kahn. "Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo." J. Cell Biol. 124, 289 - 300 (1994).

11. J.M. Lenhard, R.A. Kahn, and P.D. Stahl. "Evidence for ADP-ribosylation factor (ARF) as a regulator of in vitro endosome-endosome fusion." J. Biol. Chem. 267, 13047 - 52 (1992).

12. T. Serafini, L. Orci, M. Amherdt, M. Brunner, R.A. Kahn, and J.E. Rothman. "ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP binding protein." Cell 67, 239 - 54 (1991).

13. M.G. Waters, C.J.M. Beckers, and J.E. Rothman. "Purification of coat protomers." Methods. Enzymol. 219, 331 - 37 (1992).

14. J.G. Donaldson, D. Cassel, R.A. Kahn, and R.D. Klausner. "ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein b-COP to Golgi membranes." Proc. Natl. Acad. Sci. USA 89, 6408 - 12 (1992).

15. R.A. Kahn. "Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins." J. Biol. Chem. 266, 15595 - 97 (1991).

16. R.A. Kahn and A.G. Gilman. "The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP-binding protein." J. Biol. Chem. 261, 7906 - 11 (1986).

17. P.A. Randazzo and R.A. Kahn. "GTP hydrolysis by ADP-ribosylation factor is dependent on both an ADP-ribosylation factor GTPase-activating protein and acid phospholipids." J. Biol. Chem. 269, 10758 - 63 (1994).

18. H.A. Brown, S. Gutowski, C.R. Moomaw, C. Slaughter, and P.C. Sternweis. "ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity." Cell 75, 1137 - 44 (1993).

19. S. Cockcroft, G.M.H. Thomas, A. Fensome, B. Geny, E. Cunningham, I. Gout, et al. "Phospholipase D: a downstream effector of ARF in granulocytes." Science 263, 523 - 26 (1994).

20. S. Cockcroft. "G-protein regulated phospholipases C, D, and A2-mediated signaling in neutrophils." Biochem. Biophys. Acta 1113, 135 - 60 (1992).

21. G. Berstein, J.L. Blank, D.Y. Jhon, J.H. Exton, S. G. Rhee, and E.M. Ross. "Phospholipase C-b1 is a GTPase activating protein for Gq/11, its physiological regulator." Cell 70, 411 - 18 (1992).

22. T. Terui, R.A. Kahn, and P.A. Randazzo. "Effects of acid phospholipids on nucleotide exchange properties of ARF1: evidence for specific interactions with PIP2." J. Biol. Chem. (in press).

23. S.A. Haney and J.R. Broach. "Cdc25p, the guanine nucleotide exchange factor for the Ras proteins of Saccharomyces cerevisiae, promotes exchange by stabilizing Ras in a nucleotide-free state." J. Biol. Chem. 269, 16541 - 48 (1994).

24. P.A. Randazzo, T. Terui, S. Sturch, and R.A. Kahn. "The amino terminus of ADP-ribosylation factor 1 is essential for interaction with Gs and ARF GAP." J. Biol. Chem. (in press).

25. J.C. Amor, D.H. Harrison, R.A. Kahn, and D. Ringe. "The structure of the human ADP-ribosylation factor 1 complexed with GDP." Nature (in press).

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