Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10074-10077, September 1996 Biochemistry

Saccharomyces cerevisiae Gcsl is an ADP-ribosylation factor GTPase-activating 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 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 ) 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 transplacement in W303 strains (12). 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 (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). This mammalian that was converted to [a-32P]GDP. In the absence of Gcsl there Arf protein can functionally replace yeast Arfl and Arf2 in vivo was no spontaneous hydrolysis of Arf-bound GTP. (20), suggesting that mammalian ARF1 and the yeast Arfl/ Arf2 protein pair are functional homologs. Indeed, Gcsl also was a GAP for both yeast Arfl and Arf2 (Fig. 2). RESULTS AND DISCUSSION The gcsl-l mutation is a base-pair substitution that destroys Gcsl Activity Is Required for Efficient Secretion. Two yeast the Zn-finger motif located in the N-terminal region of Gcsl. ARF proteins, Arfl and Arf2, are members of an essential The gcsl-l mutant form of the protein expressed in E. coli protein family that has been implicated in vesicle transport: could not supply GAP activity for yeast Arfl or Arf2 (Fig. 2). cells with only the Arf2 member of this pair are partially A truncated form of Gcsl encoded by the gcsl-7 mutant gene blocked in the secretion of induced invertase (9). We assessed lacking the C-terminal third of the protein was similarly tested. the influence of Gcsl on this secretion, initially under growth Even though the gcsl-7 mutation does not affect the Zn-finger conditions that make Gcsl essential (10, 18). Stationary-phase portion of Gcsl, this truncated protein supplied only low levels gcsl v cells are unable to resume proliferation upon nutrient of GAP activity even at high protein concentrations. These in resupply at 15°C; in contrast, wild-type stationary-phase cells vitro tests establish that Gcsl is a GAP for yeast Arfl and Arf2 similarly treated promptly resume proliferation (10). Invertase and that this GAP function, like that of the mammalian ARF secretion could not be assessed, however, under these condi- GAP (7), requires an intact Zn-finger motif and C-terminal tions because even wild-type cells did not induce significant sequences. levels of invertase until cell proliferation had resumed (data Deficient Gcsl Function Causes Fluoride Sensitivity. By a not shown). We therefore assessed invertase secretion after mechanism not yet clear, fluoride inhibits the hydrolysis of resumption of cell proliferation and subsequent transfer to ARF-bound GTP, leading to an increased abundance of 15°C, a situation that allows both wild-type and gcslA mutant GTP-ARF that impairs vesicle transport and decreases growth cells to continue to proliferate (10, 18). Actively proliferating (8, 21). Decreased ARF-GAP activity, as expected for mutant wild-type and gcslA mutant cells were transferred to low- forms of Gcsl GAP, is also expected to increase the in vivo glucose growth conditions to induce invertase production (19). abundance of the GTP-ARF form because of decreased rates As shown in Fig. 1, both wild-type and gcslA mutant cells of GTP hydrolysis. Cells lacking the Arfl protein are more induced invertase upon shift to inducing conditions, but the sensitive to fluoride than are wild-type cells (8), and as shown absence of Gcsl protein impaired invertase secretion, as in Fig. 3, cells lacking Gcsl are similarly sensitized to fluoride. evidenced by the accumulation of invertase intracellularly. It is likely that decreased levels of hydrolysis of ARF-bound Downloaded by guest on October 1, 2021 10076 Biochemistry: Poon et aL Proc. Natl. Acad. Sci. USA 93 (1996) gcslA crossed with (lf I A f2A pARF2-ARFI .. Gcsl c)

au 0_) X _ r.+

E I r' Gcsl-7 FIG. 4. Genetic interactions between gcsl and arf mutations. A Gcsl-l haploid gcslA mutant strain was mated with isogenic strains PP482 or PPY30 deleted for the ARFI or ARF2 gene, respectively, and with 0.01 0.1 1 10 100 1000 strain PPY86 from which both ARFI and ARF2 had been deleted and viability maintained by a chimericARF gene in which the ARFI open protein (ng) reading frame is expressed from the ARF2 promoter (pARF2-ARFl). FIG. 2. of and mutant forms of Gcsl. GAP Resultant diploids were sporulated, and meiotic segregants were GAP activity wild-type incubated on YEPD medium at 30°C. Segregants harboring bothgcslA activity was assessed by hydrolysis of GTP bound to yeast Arfl (open symbols) and Arf2 (solid symbols) proteins. Wild-type Gcsl protein and arfA mutations, or also harboring the pARF2-ARF1 construct (Right), are indicated by arrows. GCSI segregants harboring the arflA (circles), a mutant version with an altered Zn-finger motif encoded by mutation and the pARF2-ARF1 chimeric gene at the ARF2 locus are the gcsl-l allele (ref. 10; inverted triangles), and a C-terminally indicated by asterisks. truncated version encoded by thegcsl-7 allele (ref. 10; diamonds) were assayed. regants (indicated by arrows) were severely impaired for GTP, whether caused by decreased levels of ARF protein or growth. These gcslA arflA double-mutant cells also had de- decreased ARF-GAP activity, result in increased fluoride creased growth rates in liquid culture (30°C doubling time of sensitivity. This Gcsl requirement for robust growth in fluo- 3.3 hr, compared with 2 hr for arflA single-mutant cells and 1.4 ride is consistent with an in vivo role for Gcsl in ARF- hr for gcslA single-mutant cells and wild-type cells). Presum- dependent processes. Moreover, the similar responses by cells ably the absence of Arfl can be tolerated if there is effective lacking either ARF proteins or the Gcsl ARF GAP indicate GAP activity for Arf2, whereas the absence of Gcsl can be that ARF proteins normally cycle between GTP- and GDP- tolerated if another protein provides GAP activity for Arfl bound forms in vivo. and/or Arf2. The severely impaired growth of cells lacking both Gcsl and Arfl that Gcsl Genetic Interactions Between and gcsl Mutations. To suggests provides significant arf GAP activity for Arf2 in vivo. evaluate the in vivo role of Gcsl as an ARF GAP, we took The GTP-bound form of ARF mediates the association of advantage of the ability in yeast to assess interactions by coat protein with membrane in the production of transport exploiting mutations affecting the Gcsl protein and the Arfl/ vesicles (22), whereas GAP-mediated hydrolysis of ARF- Arf2 pair. Yeast cells containing either Arfl or Arf2 protein bound GTP is thought to dissociate coatomer from vesicles in are viable (8), but cells lacking both Arfl and Arf2 are not, preparation for fusion with target membranes (23). As shown indicating that Arfl and Arf2 supply an essential function in in Fig. 5, increased levels of Gcsl GAP caused by increased a redundant fashion. A haploid gcslA mutant cell was mated GCSI gene expression had little effect on cells containing both to a haploid arflA cell that was kept alive by the Arf2 protein. Arfl and Arf2 but markedly impaired the growth of cells The resultant diploid, heterozygous for both the gcslA and lacking Arfl. The increased Gcsl abundance in these cells may arflA mutations, was then sporulated and meiotic tetrads were increase hydrolysis of GTP and so prevent the accumulation of analyzed. As shown in Fig. 4 Left, arflA gcslA& meiotic seg- Arf2-GTP to levels sufficient for vesicle function. Gcsl Provides ARF-GAP Activity in Vivo for Both Arfl and With NaF ArfM.gcslA arf2A segregants (Fig. 4 Center) grew as effectively arfliA as single-mutant or wild-type cells. To investigate whether the differences in the results observed above with arfl and arf2 mutants may stem from the different abundances of these proteins in yeast, we expressed Arfl protein at low levels by gc.slA WT replacing the open reading frame of the ARF2 gene with that ofARFI. The resultant chimericARF gene expressing the Arfl protein from the ARF2 promoter was integrated at the ARF2 locus to replace the ARF2 gene in cells already deleted for gc.slA WT ARFI. These cells, which are maintained by low-level Arfl, resembled arflA ARF2 cells in slow growth due to limiting amounts of Arf protein (Fig. 4 Right). In this situation, an increase in Gcsl abundance slowed growth even more (data not shown), indicating that the Gcsl protein can serve as a arflA GAP for Arfl to decrease the abundance of GTP-Arfl in Without NaF these mutant cells. Likewise, the absence of Gcsl in mutant FiG. 3. Fluoride sensitivity. Wild-type, gcslA, and arflA mutant cells of this type caused a further decrease in growth rate (Fig. cells (strains W303-la, PP444, and PP482, respectively) were incubated 4), presumably by limiting Arfl-GTP hydrolysis. These obser- at 30°C on YEPD solid medium with or without added NaF (40 mM) vations suggest that, both in vivo and in vitro, the Gcsl GAP as indicated. uses both Arfl and Arf2 proteins as substrate. Downloaded by guest on October 1, 2021 Biochemistry: Poon et aL Proc. Natl. Acad. Sci. USA 93 (1996) 10077 phase, whereas an alternative GAP for Arfl and/or Arf2 may Increased be more significant during exponential cell proliferation. The Control Gcs Ip availability of mutations in the Gcsl ARF GAP should facil- itate the elucidation of the role of Gcsl in vesicle transport and in cell projiferation and should provide valuable tools to identify additional GAP activities in yeast. We thank Tim Steams for plasmids; the Dalhousie yeast group for helpful discussions; and K. Gillis, A. Wheeler, and D. Carruthers for expert technical assistance. This work was supported by a grant to G.C.J. and R.A.S. from the National Cancer Institute of Canada with funds made available from the Canadian Cancer Society, and by grants to D.C. from the Israel Academy of Sciences and the Fund for Promotion of Research at the Technion. G.C.J. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada. 1. Schekman, R. & Orci, L. (1996) Science 271, 1526-1533. 2. Boman, A. L. & Kahn, R. A. (1995) Trends Biochem. Sci. 20, 147-150. 3. Donaldson, J. G., Cassel, D., Kahn, R. A. & Klausner, R. D. (1992) Proc. Natl. Acad. Sci. USA 89, 6408-6412. 4. Bednarek, S. Y., Ravazzola, M., Hosobuchi, M., Amherdt, M., Perrelet, A., Schekman, R. & Orci, L. (1995) Cell 83, 1183-1196. 5. Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E. & Mellman, I. (1995) Cell 83, 703-713. 6. Makler, V., Cukierman, E., Rotman, M., Admon, A. & Cassel, D. (1995) J. Biol. Chem. 270, 5232-5237. FIG. 5. Effect of increased expression of Gcsl in cells with 7. Cukierman, E., Huber, I., Rotman, M. & Cassel, D. (1995) decreased Arf levels. Wild-type and arflA strains (W303-la and Science 270, 1999-2002. PP482, respectively) were transformed with plasmid pPP403 carrying 8. Steams, T., Kahn, R. A., Botstein, D. & Hoyt, M. A. (1990) Mo. the GCSI gene under control of the inducible GALI,10 promoter or Cell. Biol. 10, 6690-6699. with a vector control. Cells were incubated on selective medium to 9. Steams, T., Willingham, M. C., Botstein, D. & Kahn, R. A. (1990) maintain plasmid, with galactose as the sole carbon source to induce Proc. Natl. 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Cell Bio. that this hypothesized GAP and the Gcsl GAP may have 110, 1897-1909. complementary roles. Mutant cells with insufficient Gcsl 16. Franco, M., Chardin, P., Chabre, M. & Paris, S. (1995) J. Biol. GAP function exhibit a novel cold sensitivity that is dependent Chem. 270, 1337-1341. on the physiological or growth status of the cell at the time of 17. Weiss, O., Holden, J., Rulka, C. & Kahn, R. A. (1989) J. Bio. Chem. 264, 21066-21072. transfer to the cold. This cold sensitivity (at 15°C) of gcsl 18. Drebot, M. A., Johnston, G. C. & Singer, R. A. (1987) Proc. Natl. mutant cells is only seen during the transition from the starved Acad. Sci. USA 84, 7948-7952. stationary-phase state to active cell proliferation. Stationary- 19. Carlson, M. & Botstein, D. (1982) Cell 28, 145-154. phase mutant cells stimulated to resume proliferation by 20. Kahn, R. A., Kem, F. G., Clark, J., Gelmann, E. P. & Rulka, C. addition of nutrients respond at 15°C by undergoing many of (1991) J. Bio. Chem. 266, 2606-2614. the physiological changes typical of wild-type cells (25) but, 21. Finazzi, D., Cassel, D., Donaldson, J. G. & Klausner, R. D. unlike wild-type cells, fail to resume proliferation. In marked (1994) J. Biol. Chem. 269, 13325-13330. contrast, stationary-phase gcsl mutant cells;at 30°C resume 22. Orci, L., Palmer, D. J., Amherdt, M. & Rothman, J. E. (1993) proliferation with wild-type kinetics (10). Cell physiology plays Nature (London) 364, 732-734. 23. Tanigawa, G., Orci, L., Amherdt, M. Ravazzola, M., Helms, J. B. a critical role: gcsl mutant cells first allowed to resume & Rothman, J. E. (1993) J. Cell Biol. 123, 1365-1371. proliferation at 30°C can continue to proliferate indefinitely 24. Kahn, R. A. & Gilman, A. G. (1986) J. Biol. Chem. 261, 7906- after transfer to 15°C, as long as sufficient nutrients are 7911. present. Thus the Gcsl ARF GAP has an important role 25. Werner-Washburne, M., Braun, E., Johnston, G. C. & Singer, during the resumption of cell proliferation from stationary R. A. (1993) Microbiol. Rev. 57, 383-401. Downloaded by guest on October 1, 2021