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Saccharomyces Cerevisiae Gcsl Is an ADP-Ribosylation Factor Gtpase

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Saccharomyces Cerevisiae Gcsl Is an ADP-Ribosylation Factor Gtpase Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10074-10077, September 1996 Biochemistry Saccharomyces cerevisiae Gcsl is an ADP-ribosylation factor GTPase-activating protein PAK PHI POON*, XIANGMIN WANGt, MIRIAM ROTMANt, IRIT HUBERt, EDNA CUKIERMANt, DAN CASSELt, RICHARD A. SINGERt§, AND GERALD C. JOHNSTON*¶ Departments of *Microbiology and Immunology, tBiochemistry, and §Medicine, Dalhousie University, Halifax, NS Canada B3H 4H7; and tDepartment of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel Communicated by Gregory A. Petsko, Brandeis University, Waltham, MA, June 14, 1996 (received for review April 24, 1996) ABSTRACT Movement of material between intracellular defining member of a family of proteins with structural similarity compartments takes place through the production of trans- to a recently described mammalian protein with ARF-GAP port vesicles derived from donor membranes. Vesicle budding activity (7), particularly in a conserved zinc-finger motif. that results from the interaction of cytoplasmic coat proteins In this study we have analyzed the function of Gcsl. We show (coatomer and clathrin) with intracellular organelles requires that the Gcsl protein does indeed possess ARF-GAP activity a type ofGTP-binding protein termed ADP-ribosylation factor in vitro and, furthermore, that GCcsl interacts genetically with (ARF). The GTPase cycle of ARF proteins that allows the yeast Arfl and Arf2 proteins, as assessed by effects on cell uncoating and fusion of a transport vesicle with a target growth and protein secretion. Thus Gcsl, by both in vitro and membrane is mediated by ARF-dependent GTPase-activating in vivo criteria, provides GAP activity for yeast ARF proteins. proteins (GAPs). A previously identified yeast protein, Gcsl, exhibits structural similarity to a mammalian protein with MATERIALS AND METHODS ARF-GAP activity in vitro. We show herein that the Gcsl protein also has ARF-GAP activity in vitro using two yeast Arf Strains, Plasmids, and Growth Conditions. Wild-type yeast proteins as substrates. Furthermore, Gcsl function is needed strains W303-la (MATa leu2-3, 112 ura3-1 his3-11, 15 trpl-l for the efficient secretion of invertase, as expected for a ade2-1) and the isogenic MATTa W303-lb have been described component of vesicle transport. The in vivo role of Gcsl as an (11). Strains GWK9A (10) and PP444 were constructed by ARF GAP is substantiated by genetic interactions between introducing the gcslA&::URA3 or gcslA::LEU2 allele, respec- mutations in the ARFI/ARF2 redundant pair of yeast ARF tively, by directed gene transplacement in W303 strains (12). genes and a gcsl-null mutation; cells lacking both Gcsl and Plasmids pRB1292 and pRB1296 (8) were used to disrupt the Arfl proteins are markedly impaired for growth compared ARF1 and ARF2 genes in W303-la to produce strains PP482 with cells missing either protein. Moreover, cells with de- (arflA::URA3) and PPY30 (arJ2A::LEU2), respectively. A mu- creased levels ofArfl or Arf2 protein, and thus with decreased tant strain expressing only Arfl protein but from the ARF2 levels of GTP-Arf, are markedly inhibited for growth by promoter (low-level Arfl) was derived from the arflA strain increased GCSI gene dosage, presumably because increased PP482. Using a PCR-based strategy, plasmid pPPL27 was levels of Gcsl GAP activity further decrease GTP-Arf levels. assembled carrying the ARFI ORF fused to the 5'-flanking Thus by both in vitro and in vivo criteria, Gcsl is a yeast ARF region of the ARF2 gene that includes the promoter and start GAP. codon of ARF2. The HIS3 gene was added to the 3'-flanking region to provide a selectable marker for the chimeric ARF For eukaryotic cells, movement of material between intracel- gene. The arflA strain was then transformed with a PvuII lular compartments is mediated by transport vesicles that bud restriction fragment from pPPL27, encompassing the ARF2 from a donor membrane and subsequently fuse with an promoter, the ARF1 ORF, the HIS3 gene, and ARF2 down- acceptor compartment. In the Golgi system, vesicle budding is stream sequences. Transplacement of this fragment to the driven by interactions of the cytoplasmic coat proteins termed ARF2 locus to replace the wild-typeARF2 gene was confirmed coatomer with Golgi stacks and of clathrin adaptor particles by Southern blot analysis. The ARF and GCSI status of each with the trans-Golgi network (1). Both of these processes meiotic segregant, including segregants harboring the pARF2- depend on a type of GTP-binding protein termed ADP- ARFI chimera, was determined by scoring auxotrophies. ribosylation factor 1 (ARF1) (2, 3). Members of the ARF Increased expression of the Gcsl protein from the GALJ,10 family of GTP-binding proteins may also regulate vesicle promoter was accomplished by transfer of an appropriate transport at the endoplasmic reticulum (4) and between en- restriction fragment to the multiple cloning site within the dosomes and the plasma membrane (5). The regulatory action pEMBLyex4 plasmid (12). A restriction fragment encompass- of ARF proteins, like other GTP-binding proteins, is mediated ing the GAL1,10 promoter region and the GCS1 gene was then through a GTPase cycle, which is controlled in part by transferred to the CEN-based vector pRS315 (13) to create GTPase-activating proteins (GAPs) (6, 7). plasmid pPP403. Yeast cells possess at least three ARF proteins, ofwhich two, Cells were routinely grown in standard medium (14), and and an the standard yeast genetic procedures were used (12). Invertase Arfl Arf2, constitute essential pair (8). Although secretion was assayed as described (15). proteins that regulate the GTPase cycle of ARF proteins in Bacterial Expression of Proteins. Recombinant Gcsl pro- yeast have not yet been identified, an activity that stimulates teins were expressed in Escherichia coli under control of the T7 GTP hydrolysis on Arf proteins has been detected in yeast cell promoter on plasmid pET16b (Novagen). Before transfer to extracts (9), suggesting the existence of a yeast GTPase- this GCS1 were first cloned in the activating protein for Arf proteins. On structural grounds, one expression vector, sequences candidate yeast ARF GAP is the Gcsl protein (10), the Abbreviations: ARF, ADP-ribosylation factor; GAP, GTPase- activating protein. The publication costs of this article were defrayed in part by page charge $To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Microbiology and Immunology, Dalhousie University, Halifax, NS accordance with 18 U.S.C. §1734 solely to indicate this fact. Canada B3H 4H7. e-mail: [email protected]. 10074 Downloaded by guest on October 1, 2021 Biochemistry: Poon et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10075 TrcHisC vector (Invitrogen) to position a (His)6 tag at the N terminus of Gcsl protein for subsequent protein purification (7). Similarly, the ARF1 and ARF2 genes were amplified from plasmids pRB1297 and pRB1306 (8), respectively, to incorpo- 0.3- Internal rate restriction sites for transfer to plasmid pET21b and fusion of an oligohistidine tract at the C terminus of each recombi- nant protein. Gcsl proteins were purified by Ni-NTA chromatography (Qiagen, Chatsworth, CA) according to manufacturer's in- structions (protocol 7). In brief, the pellet from 200 ml of 2- 02 v0 induced culture was suspended in 5 ml of 6 M guanidinium hydrochloride in 0.1 M sodium phosphate and 10 mM Tris HCl (pH 8), stirred for 1 hr, and centrifuged for 15 min at 10,000 x g. The supernatant was stirred for 45 min with 0.8 ml of V:4 2- ,WT Ni-NTA beads, which were then washed with the guanidinium solution, packed into a column, and washed with 8 M urea in >1.- External phosphate/Tris buffer (pH 8) and with the same urea solution brought to pH 6.3. Proteins were eluted with the urea buffer (pH 6.3) containing 0.25 mM imidazole hydrochloride. By /I gCS]A SDS/PAGE analysis, proteins were approximately 80% pure. ARF proteins were expressed at 27°C to maximize myristoyl- 0.5- ation (16). Bacterial extracts were prepared and passed through a DEAE-cellulose column as described (17). Arf proteins were subsequently purified by Ni-NTA chromatogra- phy using a 50-500 mM imidazole gradient for elution and 0 5 10 were concentrated approximately 10-fold by Centricon 30 ultrafiltration (Amicon). Time (hr) GAP Assays. GAP activity was assayed as described (6, 7). FIG. 1. Invertase secretion by wild-type and gcslA mutant cells. Briefly, recombinant myristoylated Arf proteins were pro- Wild-type cells of strain W303-la (open symbols) and isogenic gcslA duced in bacteria coexpressing Arf proteins and N- mutant cells of strain GWK9A (ref. 10; solid symbols) proliferating at myristoyltransferase (17). Bovine and yeast proteins were 15°C in medium containing 2% glucose were transferred to low- loaded with [a-32P]GTP in the presence of dimyristoyl- glucose (0.05%) medium, and at intervals cells were harvested to phosphatidylcholine and cholate, resulting in the association of assess internal and external invertase levels as described (14). 50-60% of the nucleotide with Arf proteins. Test samples were serially diluted in 25 mM Mops (pH 7.5) containing 0.1% Thus Gcsl, like Arf proteins (9), is required for efficient export hydrogenated Triton X-100 (7) and then incubated with of invertase. [a-32P]GTP-loaded Arf proteins for 15 min at 30°C. After brief Gcsl Is an ARF GAP in Vitro. To assess the role of Gcsl boiling to release the nucleotides from Arf, the nucleotides activity more directly we determined the ability of Gcsl were separated by thin-layer chromatography, and the amount protein, expressed in E. coli, to stimulate the GTPase activity of radioactivity was assayed by Phospholmager analysis. Re- of ARF proteins in vitro. Gcsl efficiently activated the GTPase sults are expressed as percentage of Arf-bound [a-32P]GTP function of rat ARF1 (7) (data not shown).
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