Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.     

A family of Arf effectors defined as suppressors of the loss of Arf function in the yeast Saccharomyces cerevisiae

Arf proteins are ubiquitous, eukaryotic regulators of virtually every step of vesicular membrane traffic. ADP-ribosylation factors are essential in yeast and the lethality resulting from either overexpression or underexpression (deletion) of Arf genes has previously been ascribed to dysregulation of the secretory process. We have identified a family of four genes (Suppressors of Arf ts, SAT) as high copy suppressors of a loss of function allele of ARF1 (arf1-3). Those proteins with SAT activity were found to contain a minimal consensus motif, including a C2C2H2 cluster with a novel and specific spacing. Genetic interactions between members of this family and with ARF1 are consistent with each sharing a common cellular pathway. Included in this family is Gcs1, a protein previously described (Poon, P. P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R. A., and Johnston, G. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10074-10077) to possess Arf GTPase-activating protein (GAP) activity, demonstrating a direct interaction between Arf and at least one of these suppressors. The suppression of the loss of Arf function by overexpression of Gcs1 and demonstration of direct, preferential binding of Gcs1 to the activated form of Arf (Arf.GTP) lead us to conclude that the biological role of Gcs1 is as an effector of the essential function of Arf in mitotic growth, rather than a down-regulator as implied by the biochemical (Arf GAP) activity. Suppression of the growth defect of arf1(-3) cells was observed under conditions that did not alter the secretory defect associated with arf1(-) mutation, indicating that the essential role of Arf in eukaryotes can be distinguished from role(s) in the secretory pathway and appear to employ distinct pathways and effectors.     

Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1)

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.     

Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15 degreesC is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER-Golgi transport.     

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.     

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.     

Nucleotide exchange on ARF mediated by yeast Gea1 protein

The ADP-ribosylation factor ARF is a small GTP-binding protein that is involved in the transport of vesicles between the endoplasmic reticulum (ER) and Golgi complex and within the Golgi complex itself. ARF cycles between inactive and membrane-associated active forms as a result of exchange of bound GDP for GTP; the GTP-bound form is an essential participant in the formation of transport vesicles. This nucleotide exchange is inhibited by the fungal metabolite brefeldin A (BFA). Here we identify a protein (Gea1) from Saccharomyces cerevisiae that is a component of a complex possessing guanine-nucleotide-exchange activity for ARF. We show that the activity of the complex is sensitive to brefeldin A and that Gea1 function is necessary for ER-Golgi transport in vivo. Gea1 contains a domain that is similar to a domain of Sec7, a protein necessary for intra-Golgi transport. We propose that Gea1 and ARNO, a human protein with a homologous Sec7 domain, are members of a new family of ARF guanine-nucleotide exchange factors.     

Dynamic recycling of ERGIC53 between the endoplasmic reticulum and the Golgi complex is disrupted by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, has recently been demonstrated to block protein transport from the endoplasmic reticulum (ER) to the Golgi complex. The ER to Golgi transport is primarily operated by the ER-Golgi intermediate compartment (ERGIC). We examined the effect of NDGA on the ERGIC, focusing on the distribution of its marker ERGIC53. In control cells ERGIC53 was distributed to vesicular tubular structures corresponding to the ERGIC as well as to the ER and the cis-Golgi, reflecting its cycling between these compartments. Upon treatment of cells with NDGA, ERGIC53 was rapidly accumulated in the Golgi and undetectable in the ER and the ERGIC. Prolonged incubation of cells with the drug, however, caused redistribution of ERGIC53 and resident Golgi proteins to the ER. Thus, it is likely that NDGA has dual effects on ERGIC53 cycling; the initial accumulation in the Golgi may be caused by blocking its retrieval from the cis-Golgi to the ER/ERGIC, while the delayed redistribution to the ER may occur through a pathway induced by the drug that is different from the COPI-dependent pathway.     

Arfaptin 1, an ARF-binding protein, inhibits phospholipase D and endoplasmic reticulum/Golgi protein transport

Class I ADP-ribosylation factors (ARFs) are essential for coatomer and clathrin coat assembly and vesicular transport in the Golgi apparatus. However, little is known about the in vivo regulation of ARF actions. Recently we cloned arfaptin 1, a 39 kDa protein that binds active, GTPgammaS-liganded ARF and translocates with it to Golgi membranes. Here we show that phorbol ester-stimulated phospholipase D (PLD) activity is inhibited in arfaptin 1-overexpressing NIH 3T3 cells and that arfaptin 1 inhibits ARF activation of Golgi-associated PLD. Since PLD activity is thought to play a role in regulating vesicular transport in the secretory pathway, we determined the rate of glycosylation of vesicular stomatitis virus glycoprotein as a measure of protein transport from the endoplasmic reticulum through the Golgi apparatus. Arfaptin 1 overexpression was found to decrease the rate of this reaction approximately two-fold. These data suggest that arfaptin 1 is a regulator of ARF action in the Golgi apparatus.     

Rab2 is essential for the maturation of pre-Golgi intermediates

The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum (ER) to the Golgi complex (Tisdale, E. J., Bourne, J. R., Khosravi-Far, R. , Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761). The Rab2 protein, like all small GTPases, contains conserved GTP-binding domains as well as hypervariable carboxyl-terminal and amino-terminal domains. While the role of the carboxyl terminus in specific membrane localization is well recognized, the potential role of the variable NH2 terminus remains to be clarified. To determine whether the NH2 terminus of Rab2 was required for its activity in vivo, a trans dominant mutant of Rab2 that inhibits ER to Golgi transport was progressively truncated and analyzed for its effect on vesicular stomatitis virus glycoprotein transport in a vaccinia-based transient expression system. Deletion of the first 14 amino-terminal residues resulted in the loss of the inhibitory properties of the mutant without affecting its post-translational processing or membrane association. To assess the potential role of the NH2 terminus in Rab2 function, a peptide corresponding to the first 13 amino acids following the initiator methionine was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the ER to the Golgi stack. This peptide was a potent inhibitor of transport. Biochemical and morphological studies revealed that the peptide strongly interfered with assembly of pre-Golgi intermediates which mediate segregation of anterograde and retrograde transported proteins en route to the Golgi. The combined results suggest that the NH2 terminus of Rab2 is required for its function and for direct interaction with components of the transport machinery involved in the maturation of pre-Golgi intermediates.     

Altered inotropic response of endothelin-1 in cardiomyocytes from rats with isoproterenol-induced cardiomyopathy

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369-377). Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.     

ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component beta-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.     

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.     

Effects of arfaptin 1 on guanine nucleotide-dependent activation of phospholipase D and cholera toxin by ADP-ribosylation factor

Arfaptin 1, a approximately 39-kDa protein based on the deduced amino acid sequence, had been initially identified in a yeast two-hybrid screen using dominant active ARF3 (Q71L) as bait with an HL-60 cDNA library. It was suggested that arfaptin 1 may be involved in Golgi functions, since the FLAG-tagged protein was associated with Golgi membranes when expressed in COS-7 cells and could be bound to Golgi in vitro in an ADP-ribosylation factor (ARF)- and GTPgammaS-dependent, brefeldin A-inhibited fashion. Arfaptin 2, found in the same two-hybrid screen as arfaptin 1, is 60% identical in amino acid sequence and may or may not have an analogous function. We now report some effects of arfaptin 1 on ARF activation of phospholipase D and cholera toxin ADP-ribosyltransferase. Arfaptin 1 inhibited activation of both enzymes in a concentration-dependent manner and was without effect in the absence of ARF. Two ARF1 mutants that activated the toxin, one lacking 13 N-terminal amino acids and the other, in which 73 residues at the N terminus were replaced with the analogous sequence from ARL1, were not inhibited by arfaptin, consistent with the conclusion that arfaptin interaction requires the N terminus of ARF. This region has also been implicated in phospholipase D activation, but whether the two proteins interact with the same structural elements in ARF remains to be determined. Arfaptin inhibition of the action of ARF5 and ARF6 was less than that of ARF1 and ARF3; its effects were less on nonmyristoylated than myristoylated ARFs. Arfaptin effects on guanine nucleotide binding by ARFs were minimal whether or not a purified ARF guanine nucleotide-exchange protein was present. These findings indicate that arfaptin acts as an inhibitor of ARF actions in vitro, raising the possibility that it has a similar role in vivo.     

Overexpression and functional analysis of a mitogen-inducible nuclear GTPase activating protein, Spa-1

The two Ras-related GTPases called Rap1 and Rsr1, which share 50% sequence identity with Ras GTPases are known to be activated by two distinct mammalian GAPs, i.e. cytosolic GAP3c of 55 kDa and membrane-bound GAP3m of 85 kDa. Recently we have cloned a gene encoding a 68 kDa (p68) protein product, which is associated with chromosomes during interphase. The N-terminal 190 amino acids share 43% sequence identity with the second half of the GTPase activating domain (residues 210-397) of GAP3m. The N-terminal fragment of 209 amino acids of Spa-1 (called Span-N) was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein and affinity purified. Rap1 and Rsr1 GTPase stimulatory activity of Spa-1 was tested and compared with GAP3m. Spa-1 preferentially stimulates Rsr1 GTPase rather than Rap1 GTPase, while GAP3m has a preference for Rap1 GTPase. This suggests that although Spa-1 and GAP3m stimulate GTPase of Rap1 family members, they differ in affinity for them. By mutational analysis it was also found that amino acid residues 10-183 are enough for Rap GAP activity of Spa-1.     

Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.     

Cloning and characterization of a rhoGAP homolog from Dictyostelium discoideum

Small GTPases interact with a variety of proteins that affect nucleotide binding and cleavage. GTPase activating proteins (GAPs) are one class of these proteins that act by accelerating the intrinsic GTPase rate resulting in the formation of the biologically inactive GDP-bound form of the GTPase. For the Rho subfamily of GTPases, there is a growing number of proteins with rhoGAP activity that are identifiable by a homologous region of about 150 amino acids. We have exploited this homology using the polymerase chain reaction to clone the first rhoGAP homolog, called DdRacGAP, from the slime mold Dictyostelium discoideum. The GAP domain of DdRacGAP (amino acids 1-212), when expressed and purified from Escherichia coli, is active on both Dictyostelium and human Rho family GTPases but not human Ras. The full-length protein is 1356 amino acids in length and has several interesting homologies in addition to the GAP domain, including an SH3 domain, a dbl homology domain, and a pleckstrin homology domain.     

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

ARF is required for maintenance of yeast Golgi and endosome structure and function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Delta, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4-64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4-64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.     

Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A.