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IDENTIVnCATmN OF TRANSPORT INTERMEDIATES
[34]
pooled. This step gives approximately 24-fold purification with excellent recovery of GTPyS-binding activity in a volume of 8 ml (see Table I). The pooled fractions are then diluted with an equal volume of TMD buffer to give a final concentration of 50 m M NaC1. This is loaded onto a 12-ml DEAE-Sephacel column (2.5 × 2.5 cm) equilibrated with TMD buffer containing 50 m M NaC1. The column is washed with the same buffer until the absorbance at 280 nm returns to baseline. The peak of GTP~,S-binding activity is pooled and concentrated approximately 18-fold to 1.5 ml by pressure filtration in a stirred cell containing an Amicon PM 10 membrane. The protein is then rapidly frozen and stored at - 80 o. The overall purification is approximately 34-fold (see Table I), with a yield of about 500/tg of active protein and a purity of approximately 80-90% as analyzed by SDS-PAGE and Coomassie Brilliant blue staining of the gel. The GDP off-rate, GTP on-rate, and intrinsic GTP hydrolysis rate of the bacterially produced Sec4p are quite similar to those found for Sec4p isolated from yeast. Acknowledgments This work was supported by Grants GM35370 and CA46218 to P.N. from the National Institutes of Health. M.G. was supported by the Lucille P. Markey Charitable Trust, P.B. was supported by the Damon Runyon Walter Winchell Cancer Research Fund, and A.K.K. was supported by the Jane Coffin Childs Memorial Fund for Medical Research and by a Swebilius Cancer Research Award.
[34] P r e p a r a t i o n o f R e c o m b i n a n t ADP-Ribosylation Factor
By PAUL A. RANDAZZO, OFRA WEISS, and RICHARD A. KArIN Introduction ADP-ribosylation factor (ARP0 proteins were originally identified and purified based on an in vitro activity as the protein cofactor required for efficient ADP-ribosylation of Gs by cholera toxin ~ (for recent reviews see Kahn3,4). Subsequently, ARF was shown to be a 21-kDa GTP-binding t L. S. Schleifer, R. A. Kahn, E. Hansld, J. K. Northup, P. C. Sternweis, and A. G. Gilman, J. Biol. Chem. 257, 20 (1982). 2 R. A. Kahn and A. G. Gilman, J. Biol. Chem. 259, 6228 (1984). R. A. Kahn, in "G Proteins" (L. Birnbaumer and R. Iyengar, eds.), p. 201. Academic Press, Orlando, Florida, 1990. 4 R. A. Kahn, this series, Vol. 195, p. 233.
METHODSIN ENZYMOLOGY,VOL 219
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[34]
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protein that is active only in the GTP-bound form. 5 ADP-ribosylation factor activity and/or immunoreactivity have been found in every eukaryotic cell tested but is absent from Escherichia coli. 3 Multiple genes for ARF have been found in every cell type examined, including two homologous genes for ARF in the yeast Saccharomyces cerevisiae, 6 two identified in bovine cDNA libraries, 7,s and three found in human cDNA libraries. 9- H A large number of ARF and ARF-like cDNAs have been identifiedn and have led to the establishment of ARF and related proteins as the fourth subfamily of the RAS superfamily of low molecular weight GTP-binding proteins. Hallmarks for this subfamily of proteins include myristoylation at the amino-terminal glycine instead of carboxy-terminal processing (common to the other three subfamilies in the RAS superfamily) and specific protein sequence motifs, e.g., D(V/I)GGQ instead of DTAGQ, as found in the other three subfamilies at the second consensus GTP-binding domain) ADP-ribosylation factor is also highly functionally conserved, z~ and can be distinguished from other GTP-binding proteins, including the ARF-like proteins, ~a by two criteria that define a bona fide ARF from a structurally related protein. The ARF proteins have activity in the ARF assay, as cofactor for cholera toxin-dependent ADP-ribosylation, tt and expression of an ARF protein can rescue the lethal double-mutant arfl-arf2- in yeast, n Examples of ARF-related genes include arP 3 from Drosophila melanogaster and S A R P 4 and CIN4 ~5 found in S. cerevisiae. Each of these gene products are in the ARF subfamily of proteins but lack ARF activities, as defined above. We will limit further discussion to activities found in association with these functionally defined ARF proteins. ADP-ribosylation factor has been implicated as a critical component in the protein secretory machinery in yeast (S. cerevisiae) and mammalian cells. Disruption of the ARF1 gene in yeast causes a defect in N-glycosyla5 R. A. Kahn and A. G. Gilman, J. Biol. Chem. 261, 7906 (1986). 6 T. Stearns, R. A. Kahn, D. Botstcin, and M. A. Hoyt, Mol. Cell. Biol. 10, 6690 (1990). 7 j. Scwcll and R. A. Kahn, Proc. Natl. Acad. Sci. USA 85, 4620 (1988). 8 S. R. Price, M. Nightingale, S. C. Tsai, K. C. Williamson, R. Adamik, H. C. Chert, J. Moss, and M. Vaughan, Proc. Natl. Acad. Sci. USA 85, 5488 (1988). 9 Z. Peng, I. Calver, J. Clark, L. Helman, R. A. Kahn, and H. Kung, BioFactors 2, 45 (1989). zo D. A. Bobak, M. S. Nightingale, J. J. Mu~_n£h~S. R. Price, J. Moss, and M. Vanghan, Proc. Natl. Acad. Sci. USA 86, 6101 (1989). " R. A. Kahn, F. G. Kern, J. Clark, E. P. Crelman, and C. Rulka, J. Biol. Chem. 266, 2606 (1991). ~2j. Clark and R. A. Kahn, unpublished observations, 1990. t3 j. W. Tamkun, R. A. Kahn, M. Kissinger, B. J. Brizuela, C. Rulka, M. P. Scott, and J. A. Kennison, Proc. Natl. Acad. Sci. USA 88, 3120 (1991). 1, A. Nakano and M. Muramatsu, J. CellBiol. 109, 2677 (1989). LsT. Stearns, R. A. Kahn, M. A. Hoyt, and D. Botstein, in preparation.
364
IDENTIFICATION OF T R A N S P O R T INTERMEDIATES
[34]
tion, similar to that seen for y p t l mutants, as evidenced by the formation of incompletely glycosylated invertase. 16 Furthermore, the arfl- mutant shows synthetic lethality with yptl-1, sec21-1, and bet2-1, each of which has been shown to cause defects in protein secretion. ~6 In NIH 3T3, ~6 Chinese hamster ovary (CHO), 17 and normal rat kidney (NRK) cells, ~s immunocytochemical techniques have allowed the localization of ARF to Golgi membranes and Golgi-derived vesicles. In fact, ARF has been shown to be an abundant protein on non-clathrin-coated vesicles. '7 The binding of ARF proteins and fl-COP to Golgi membranes has been found to be rapidly and specifically blocked by addition ofbrefeldin A, both in vivo and in vitro. TM This has been taken as support for a model for ARF action involving cycling between soluble and particulate pools, similar to the model proposed for SEC4.19 It has proved difficult to demonstrate definitively a requirement for ARF proteins in a specific step of the protein secretory pathway as ARF is a ubiquitous and abundant protein, present in both cytosol and membrane preparations. None of the antibodies currently available have neutralizing properties or the ability to immunoprecipitate the native proteins. The discovery of potent and specific peptide inhibitors of ARF activities has allowed the demonstration of an absolute requirement for ARF in in vitro secretion assays2°,2~ and dearly shown a role for ARF proteins in secretion. Peptides derived from the N terminus of either m A R F I p or mARF4p inhibited ARF association with a Golgi membrane fraction from CHO cells) 2 blocked vesicle budding from a similar membrane preparation, 2° and blocked ER-to-Golgi 2~ and intra-Golgi transport, 2° measured in vitro using CHO cellular fractions. Although a role for ARF in protein secretion appears established, much more work is needed to clarify the roles of GTP binding and hydrolysis by ARF as a regulatory event in secretion, the specific step which requires ARF (e.g., budding, fusion, etc.), and the site of action of brefeldin A, and to answer many more questions of interest to both workers in the protein secretion field as 16T. Stearns, M. C. Willingham, D. Botstein, and R. A. Kahn, Proc. Natl. Acad. Sci. USA 87, 1238 (1990). 17T. Serafini, L. Orci, M. Amherdt, M. Brunner, R. A. Kahn, and J. E. Rothman, Cell 67, 239 (1991). ~8j. Donaldson, R. A. Kahn, J. Lippincott-Schwartz,and R. D. Klausner, Science 254, 1197 (1991). ~9 N. C. Walworth, B. Goud, A. Kastan-Kabcenell, and P. J. Novick, EMBO J. 8, 1685 (1989). 2oR. A. Kahn, P. Randazzo, T. Serafini, O. Weiss,C. Rulka, J. Clark, M. Amherdt, P. Roller, L. Orci, J. E. Rothman, J. Biol. Chbm. 267, 13039(1992). 21W. Balch, R. A. Kahn, and R. Schwaninger,J. Biol. Chem. 267, 13053(1992). 22j. G. Donaldson, D, Cassel, R. A. Kahn, and R. D. Klausner, Proc. Natl./lead. Sei. USA in press (1992).
[34]
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well as those elucidating mechanisms for members of the RAS supeffamily of proteins. In all tissues thus far examined, ARF is purified as a closely spaced doublet containing an unknown number of gcne products.2,9 The use of a single gene product is desirable for studies aimed at elucidating the role(s) of individual proteins in a specific biochemical reaction, for example examining the role of ARF in secretion. Thus, rather than using ARFs purified from mammalian or yeast cells we have made use of the expression system of Studier and Moffatt23 to overproduce specific A R F gene products for use in biochemical studies of protein function. The recombinant protein is indistinguishable from that purified from tissues in terms of nucleotide-binding kinetics and activity in cholera toxin-catalyzed ADPribosylation of Gs.24 The only known covalent modification to ARF proteins is N-terminal myristoylation,3 which is essential for function in vivo but has no effect on activity in the ARF assay. While bacteria are incapable of protein N-myristoylation, it has been shown that coinduction of both the protein of interest and the yeast myristoyl-CoA: protein N-myristoyltransferase (NMT) results in the formation of properly myfistoylated proteins in bacteria. 25 As we begin to reconstitute recombinant ARF proteins into more crude systems that allow studies of function it is clear that having both myristoylated and nonmyristoylated forms of the protein is useful. The methods used in obtaining both nonmyristoylated and myristoylated ARF proteins are described below.
Methods The methods described below allow the expression and purification of ARF proteins in bacteria. Typically, ARF can constitute as much as 10% of total bacterial protein, which results in a yield of 1-5 mg of purified ARF from I liter of cells. ADP-ribosylation factor can be monitored during purification and quantitated using a radioligand binding assay described by Kahn and Gilman, 5 a modification of the method described by Northup et al. 26 The best estimate of final purity is obtained from densitometric scanning of stained sodium dodecyl sulfate (SDS) gels or quantitation of protein-associated GDP. 5 23 F. W. Studier and B. A. Moffatt, J. Mol. Biol. 189, 113 (1986). 24 O. Weiss, J, Holden, C. Rulka, and R. A. Kahn, J. Biol. Chem. 264, 21066 (1989). 25 R. J. Duronio, E. Jackson-Machelski, R. O. Heuckeroth, P. O. Olins, C. S. Devine, W. Yonemoto, L. W. Slice, S. S. Taylor, and J. I. Gordon, Proc. Natl. Acad, Sci. USA 87, 1506 (1990). 26 j. K. Northup, M. D. Smigel, and A. G. Gilman, J. Biol. Chem. 257, 11416 (1982).
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IDENTIFICATION OF TRANSPORT INTERMEDIATES
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Protein Expression High-level expression of several ARF and ARF-related proteins has been achieved by using the T7 polymerase/promoter system described by Studier et a/. 23,27 The construction of the ARF expression plasmid, pOW12, has been described previously.24 Briefly, this entails insertion of the coding region of the gene into the pET3C vector~3,27 such that the initiating methionine is part of an NdeI restriction site that is located at the optimal distance from a T7 promoter to direct maximal expression. BL2 I(DE3) cells27 contain the T7 polymerase structural gene under control of the lacUV5 promoter and is thus induced by isopropyl-fl-othiogalactopyranoside (IPTG). BL21 (DE3) cells transformed with pOW 12 are grown in LB medium containing 100/tg/ml ampicillin at 37 °. When the A~0o~- 1.0, IPTG is added to a final concentration of 1 mM. Ninety minutes later, the cells are harvested by centrifugation at 4000 g for 15 min at 4 ° and may be stored at - 8 0 °.
Purification Purification of the recombinant protein is accomplished in two steps, batch elution from DEAE-Sephacel and a gel-filtration column. This protocol routinely results in a product that is at least 85% pure and has been used successfully for recombinant human A R F I I ~ and ARF4p, II drosophila ARL lp, 13and wild-type and eight point mutants of yeast ARFI p.2S 1. The cell pellet from a l-liter culture is resuspended in 20 ml of 50 m M Tris-HC 1, pH 8.0, 40 m M ethylenediaminetetraacetic acid (EDTA), 25% (w/v) sucrose, I mg/ml lysozyme and incubated at room temperature for 15 min. The cells are lysed by the addition of 8 ml of 0.2% (w/v) Triton X-100, 50 mMTris-HC1, pH 8.0, 100 m M MgC12. After l0 rain at 4 °, t h e suspension is clarified by centrifugation at 100,000-g for 60 rain at 4*. 2. The supernatant is loaded onto a 50-ml DEAE-Sephacel column, previously equilibrated in three column volumes of 20 m_MTris-HC1, pH 7.4, 1 m M EDTA, 1 m M dithiothreitol, 50 m M NaC1. The flow through and a wash of one column volume are collected and concentrated to 4 ml by ultrafiltration using an Amicon (Danvers, MA) YM 10 membrane. 3. The concentrate is applied to an Ultrogel AcA 54 column (200 ml, 40 X 2.5 cm; IBF Biotechnics, Columbia, MD) that has been equilibrated with 20 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, 1 m M EDTA, 100 m M NaCl, 1 m M dithiothreitol 27 A. H. Rosenberg, B. N. Lade, D. Chui, S. W. Lin, J. J. Dunn, and F. W. Studier, Gene 56, 125 (1987). 28 R. A. Kahn, J. Clark, and C. Rulka, unpublished observations, 1990.
[34]
RECOMBINANT ADP-RIBOSYLATION FACTOR
367
(DTT), and 2 m M MgC12. The column is developed in the same buffer at a flow rate of 20 ml/hr and 2.5-ml fractions are collected. The Coomassie blue-stained polyacrylamide gels of protein from each step of the purification are shown in Fig 1. The fractions containing pure ARF are pooled and concentrated by ultrafiltration to I mg/ml and stored at - 80 °.
GTP-BindingAssay ADP-ribosylation factor is easily detected by nucleotide binding and this is the method used in our laboratory for monitoring the elution of recombinant ARF from chromatographic columns during purification. As previously described,5 nucleotide exchange on ARF requires phospholipid and detergent and is most efficient at low concentrations of Mg2+. Hence, the binding cocktail contains 25 m M HEPES, pH 7.4-8.0, with
68
---,"
4 3 ---,"
29
"-
18-"-'-
1
2
3
4
5
FIG. 1. Purification of mARFlp from bacteria. Coomassie blue stained-SDS polyacrylamide gel fractionation of proteins from each step of the purification of mARFlp from BL21 (DE3) cells transfected with pOW12. Lane l, total cell extract from cells induced with IPTG; lane 2, pooled DEAE-Sephacel fractions; lane 3, pooled Ultrogel AcA 54 fractions (purified recombinant mARFlp); lane 4, pooled Ultrogel AcA 54 fractions from bacteria transfected with expression vectors for mARF1 and N-myristoyltransferase; lane 5, ARF purified from bovine brain as previously described.4 Molecular weight markers ( X 10-s) on left-hand side.
368
IDENTIFICATION OF TRANSPORT INTERMEDIATES
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100 mMNaC1, 1 m M EDTA, 0.5 mMMgCI2, 1 m M D T T , 3 m M L-c~dimyristoylphosphatidylcholine(DMPC), 0.1% (w/v) sodium cholate, and 1 a M [ot-32p]GDP (I 0,000 cpm/pmol), [y-32p]GTP (10,000 cpm/pmol), or [35S]GTPyS (10,000 cpm/pmol).4,5 The DMPC is freshly prepared by sonication of a 30 m M solution in 20 m M HEPES, 1 m M EDTA, and 2 m M M g C I 2 immediately prior to performing the binding assay. The turbid suspension is sonicated until it becomes translucent and then mainmined at 30 ° until diluted into the binding cocktail. The protein is incubated in the binding cocktail for 1 hr at 30 ° in a total volume of 50/~1 and exchange is stopped by the addition of ice-cold TNMD buffer: 2 ml of Tris, pH 7.4, 100 mMNaC1, 10 mMMgC12, and 1 m M D T T . The bound and free nucleotides are separated by filtration through 25-mm BA85 nitrocellulose filters (Schleicher & Schuell, Keene, NH). The filters are washed six times with 2 ml ice-cold TNMD. ADP-ribosylation factor-nucleotide complex is retained and can be quantified by scintillation spectroscopy. Binding typically increases linearly for the first 30 min and reaches a plateau within 1 to 3 hr. Binding stoichiometries tend to be poor, especially for triphosphates and triphosphate analogs, as the protein contains 1 mol bound GDP/mol protein. As GDP has at least a 100-fold greater affinity for the protein than the triphosphates the stoichiometry of GTP binding is no more than 0.1. Hence, ARF is not easily quantified by this assay.
Quantitation Typically, the ARF prepared by the protocol above is 85% pure (Fig. 1) which can be assessed by Coomassie blue staining of protein fractionated on polyacrylamide gels. As active ARF is purified as a 1 : 1 complex with GDP, ~ the concentration of active ARF can also be assessed by determining the amount of nucleotide released after heat denaturation of a given amount of the purified protein. 5,24
Comments 1. The time of induction can be varied. With some proteins, accumulation continues beyond 90 min and yields are improved with longer (3-4 hr) inductions. 2. Batch elution from DEAE-Sephacel is the most variable step. Elution with higher concentrations of NaCI increases the yield of ARF but generally results in a less pure preparation after gel filtration. A significant fraction of ARF (as much as 50%) is also found in the 100,000 g pellet. This has been purified in the presence of 7 M urea and results in a nucleotide-free preparation of ARF that is much less stable24 than ARF purified by the protocol presented in this chapter.
[35]
Ypt PROTEINSIN YEAST
369
3. This approach has provided our laboratory with purified recombinant ARF proteins from several sources. It has also been modified to obtain myristoylated recombinant proteins. In this case, the bacteria are cotransfected with the plasmid containing the ARF coding region as well as pBB131, 25 a plasmid containing the yeast N-myristoyltransferase gene N M T 1 under the same T7 promoter,25 and a marker for kanamycin resistance to allow selection for both plasmids. The BL21(DE3) cells are then grown in LB medium containing 50/~g/ml kanamycin and 100/zg/ml ampicillin. On induction with IPTG we also add 200 # M myristic acid [125 m M stock in 50% (v/v) methanol is freshly prepared]. Purification, GTP-binding determination, and quantitation are all performed as for the nonmyristoylated protein. Using this approach, approximately 10-60% of the ARF is myristoylated as determined by sequencing of the amino terminus of the purified proteins.28 The myristoylated protein cannot be separated from the unmodified protein using ion-exchange, gel-filtration, or hydrophobic interaction chromatography, including phenyl-Sepharose or heptylamine-Sepharose.28 As is true for the nonmyristoylated recombinant protein, the myristoylated recombinant protein is indistinguishable from ARF purified from bovine brain in terms of nucleotide binding kinetics and activity in the cholera toxin-catalyzed reaction. 2s
[35] By P E T E R
Ypt Proteins in Yeast
WAGNER, LUDGER HENGST,
and
DIETER GALLWITZ
Introduction The ras superfamily of genes encodes structurally related, guanine nucleotide-binding proteins of similar size (around 200 amino acid residues) and biochemical properties. 1-3 From work with yeast mutants, it is evident that members of the Ypt subfamily of proteins,4 including Ypt 1
t H. R. Bourne, D. A. Sanders, and F. McCormick, Nature (London) 348, 125 (1990). 2 H. R. Bourne, D. A. Sanders, and F. McCormick, Nature (London) 349, 117 (1991). 3 A. Hall, Science 249, 635 (1990). 4 D. Gallwitz, H. Haubruck, C. Molenaar, R. Pran__ge,M. Puzieha, H. D. Sehmitt, C. Vorgias, and P. Wagner, in "The Guanine-Nucleotide Binding Proteins" (L. Bosch, B. Kraal, and A. Parmeggiani, eds.), NATO ASI Ser. A, Vol. 165, p. 257. Plenum, New York, 1989.
METHODS IN ENZYMOLOGY, VOL. 219
Copyright© 1992by AcademicPress,Inc. All rightsof reproductionin any formreserved.
IDENTIVnCATmN OF TRANSPORT INTERMEDIATES
[34]
pooled. This step gives approximately 24-fold purification with excellent recovery of GTPyS-binding activity in a volume of 8 ml (see Table I). The pooled fractions are then diluted with an equal volume of TMD buffer to give a final concentration of 50 m M NaC1. This is loaded onto a 12-ml DEAE-Sephacel column (2.5 × 2.5 cm) equilibrated with TMD buffer containing 50 m M NaC1. The column is washed with the same buffer until the absorbance at 280 nm returns to baseline. The peak of GTP~,S-binding activity is pooled and concentrated approximately 18-fold to 1.5 ml by pressure filtration in a stirred cell containing an Amicon PM 10 membrane. The protein is then rapidly frozen and stored at - 80 o. The overall purification is approximately 34-fold (see Table I), with a yield of about 500/tg of active protein and a purity of approximately 80-90% as analyzed by SDS-PAGE and Coomassie Brilliant blue staining of the gel. The GDP off-rate, GTP on-rate, and intrinsic GTP hydrolysis rate of the bacterially produced Sec4p are quite similar to those found for Sec4p isolated from yeast. Acknowledgments This work was supported by Grants GM35370 and CA46218 to P.N. from the National Institutes of Health. M.G. was supported by the Lucille P. Markey Charitable Trust, P.B. was supported by the Damon Runyon Walter Winchell Cancer Research Fund, and A.K.K. was supported by the Jane Coffin Childs Memorial Fund for Medical Research and by a Swebilius Cancer Research Award.
[34] P r e p a r a t i o n o f R e c o m b i n a n t ADP-Ribosylation Factor
By PAUL A. RANDAZZO, OFRA WEISS, and RICHARD A. KArIN Introduction ADP-ribosylation factor (ARP0 proteins were originally identified and purified based on an in vitro activity as the protein cofactor required for efficient ADP-ribosylation of Gs by cholera toxin ~ (for recent reviews see Kahn3,4). Subsequently, ARF was shown to be a 21-kDa GTP-binding t L. S. Schleifer, R. A. Kahn, E. Hansld, J. K. Northup, P. C. Sternweis, and A. G. Gilman, J. Biol. Chem. 257, 20 (1982). 2 R. A. Kahn and A. G. Gilman, J. Biol. Chem. 259, 6228 (1984). R. A. Kahn, in "G Proteins" (L. Birnbaumer and R. Iyengar, eds.), p. 201. Academic Press, Orlando, Florida, 1990. 4 R. A. Kahn, this series, Vol. 195, p. 233.
METHODSIN ENZYMOLOGY,VOL 219
Copyright© 1992by AcademicPress,Inc. Allrightsof reproduction in any formreserved.
[34]
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protein that is active only in the GTP-bound form. 5 ADP-ribosylation factor activity and/or immunoreactivity have been found in every eukaryotic cell tested but is absent from Escherichia coli. 3 Multiple genes for ARF have been found in every cell type examined, including two homologous genes for ARF in the yeast Saccharomyces cerevisiae, 6 two identified in bovine cDNA libraries, 7,s and three found in human cDNA libraries. 9- H A large number of ARF and ARF-like cDNAs have been identifiedn and have led to the establishment of ARF and related proteins as the fourth subfamily of the RAS superfamily of low molecular weight GTP-binding proteins. Hallmarks for this subfamily of proteins include myristoylation at the amino-terminal glycine instead of carboxy-terminal processing (common to the other three subfamilies in the RAS superfamily) and specific protein sequence motifs, e.g., D(V/I)GGQ instead of DTAGQ, as found in the other three subfamilies at the second consensus GTP-binding domain) ADP-ribosylation factor is also highly functionally conserved, z~ and can be distinguished from other GTP-binding proteins, including the ARF-like proteins, ~a by two criteria that define a bona fide ARF from a structurally related protein. The ARF proteins have activity in the ARF assay, as cofactor for cholera toxin-dependent ADP-ribosylation, tt and expression of an ARF protein can rescue the lethal double-mutant arfl-arf2- in yeast, n Examples of ARF-related genes include arP 3 from Drosophila melanogaster and S A R P 4 and CIN4 ~5 found in S. cerevisiae. Each of these gene products are in the ARF subfamily of proteins but lack ARF activities, as defined above. We will limit further discussion to activities found in association with these functionally defined ARF proteins. ADP-ribosylation factor has been implicated as a critical component in the protein secretory machinery in yeast (S. cerevisiae) and mammalian cells. Disruption of the ARF1 gene in yeast causes a defect in N-glycosyla5 R. A. Kahn and A. G. Gilman, J. Biol. Chem. 261, 7906 (1986). 6 T. Stearns, R. A. Kahn, D. Botstcin, and M. A. Hoyt, Mol. Cell. Biol. 10, 6690 (1990). 7 j. Scwcll and R. A. Kahn, Proc. Natl. Acad. Sci. USA 85, 4620 (1988). 8 S. R. Price, M. Nightingale, S. C. Tsai, K. C. Williamson, R. Adamik, H. C. Chert, J. Moss, and M. Vaughan, Proc. Natl. Acad. Sci. USA 85, 5488 (1988). 9 Z. Peng, I. Calver, J. Clark, L. Helman, R. A. Kahn, and H. Kung, BioFactors 2, 45 (1989). zo D. A. Bobak, M. S. Nightingale, J. J. Mu~_n£h~S. R. Price, J. Moss, and M. Vanghan, Proc. Natl. Acad. Sci. USA 86, 6101 (1989). " R. A. Kahn, F. G. Kern, J. Clark, E. P. Crelman, and C. Rulka, J. Biol. Chem. 266, 2606 (1991). ~2j. Clark and R. A. Kahn, unpublished observations, 1990. t3 j. W. Tamkun, R. A. Kahn, M. Kissinger, B. J. Brizuela, C. Rulka, M. P. Scott, and J. A. Kennison, Proc. Natl. Acad. Sci. USA 88, 3120 (1991). 1, A. Nakano and M. Muramatsu, J. CellBiol. 109, 2677 (1989). LsT. Stearns, R. A. Kahn, M. A. Hoyt, and D. Botstein, in preparation.
364
IDENTIFICATION OF T R A N S P O R T INTERMEDIATES
[34]
tion, similar to that seen for y p t l mutants, as evidenced by the formation of incompletely glycosylated invertase. 16 Furthermore, the arfl- mutant shows synthetic lethality with yptl-1, sec21-1, and bet2-1, each of which has been shown to cause defects in protein secretion. ~6 In NIH 3T3, ~6 Chinese hamster ovary (CHO), 17 and normal rat kidney (NRK) cells, ~s immunocytochemical techniques have allowed the localization of ARF to Golgi membranes and Golgi-derived vesicles. In fact, ARF has been shown to be an abundant protein on non-clathrin-coated vesicles. '7 The binding of ARF proteins and fl-COP to Golgi membranes has been found to be rapidly and specifically blocked by addition ofbrefeldin A, both in vivo and in vitro. TM This has been taken as support for a model for ARF action involving cycling between soluble and particulate pools, similar to the model proposed for SEC4.19 It has proved difficult to demonstrate definitively a requirement for ARF proteins in a specific step of the protein secretory pathway as ARF is a ubiquitous and abundant protein, present in both cytosol and membrane preparations. None of the antibodies currently available have neutralizing properties or the ability to immunoprecipitate the native proteins. The discovery of potent and specific peptide inhibitors of ARF activities has allowed the demonstration of an absolute requirement for ARF in in vitro secretion assays2°,2~ and dearly shown a role for ARF proteins in secretion. Peptides derived from the N terminus of either m A R F I p or mARF4p inhibited ARF association with a Golgi membrane fraction from CHO cells) 2 blocked vesicle budding from a similar membrane preparation, 2° and blocked ER-to-Golgi 2~ and intra-Golgi transport, 2° measured in vitro using CHO cellular fractions. Although a role for ARF in protein secretion appears established, much more work is needed to clarify the roles of GTP binding and hydrolysis by ARF as a regulatory event in secretion, the specific step which requires ARF (e.g., budding, fusion, etc.), and the site of action of brefeldin A, and to answer many more questions of interest to both workers in the protein secretion field as 16T. Stearns, M. C. Willingham, D. Botstein, and R. A. Kahn, Proc. Natl. Acad. Sci. USA 87, 1238 (1990). 17T. Serafini, L. Orci, M. Amherdt, M. Brunner, R. A. Kahn, and J. E. Rothman, Cell 67, 239 (1991). ~8j. Donaldson, R. A. Kahn, J. Lippincott-Schwartz,and R. D. Klausner, Science 254, 1197 (1991). ~9 N. C. Walworth, B. Goud, A. Kastan-Kabcenell, and P. J. Novick, EMBO J. 8, 1685 (1989). 2oR. A. Kahn, P. Randazzo, T. Serafini, O. Weiss,C. Rulka, J. Clark, M. Amherdt, P. Roller, L. Orci, J. E. Rothman, J. Biol. Chbm. 267, 13039(1992). 21W. Balch, R. A. Kahn, and R. Schwaninger,J. Biol. Chem. 267, 13053(1992). 22j. G. Donaldson, D, Cassel, R. A. Kahn, and R. D. Klausner, Proc. Natl./lead. Sei. USA in press (1992).
[34]
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well as those elucidating mechanisms for members of the RAS supeffamily of proteins. In all tissues thus far examined, ARF is purified as a closely spaced doublet containing an unknown number of gcne products.2,9 The use of a single gene product is desirable for studies aimed at elucidating the role(s) of individual proteins in a specific biochemical reaction, for example examining the role of ARF in secretion. Thus, rather than using ARFs purified from mammalian or yeast cells we have made use of the expression system of Studier and Moffatt23 to overproduce specific A R F gene products for use in biochemical studies of protein function. The recombinant protein is indistinguishable from that purified from tissues in terms of nucleotide-binding kinetics and activity in cholera toxin-catalyzed ADPribosylation of Gs.24 The only known covalent modification to ARF proteins is N-terminal myristoylation,3 which is essential for function in vivo but has no effect on activity in the ARF assay. While bacteria are incapable of protein N-myristoylation, it has been shown that coinduction of both the protein of interest and the yeast myristoyl-CoA: protein N-myristoyltransferase (NMT) results in the formation of properly myfistoylated proteins in bacteria. 25 As we begin to reconstitute recombinant ARF proteins into more crude systems that allow studies of function it is clear that having both myristoylated and nonmyristoylated forms of the protein is useful. The methods used in obtaining both nonmyristoylated and myristoylated ARF proteins are described below.
Methods The methods described below allow the expression and purification of ARF proteins in bacteria. Typically, ARF can constitute as much as 10% of total bacterial protein, which results in a yield of 1-5 mg of purified ARF from I liter of cells. ADP-ribosylation factor can be monitored during purification and quantitated using a radioligand binding assay described by Kahn and Gilman, 5 a modification of the method described by Northup et al. 26 The best estimate of final purity is obtained from densitometric scanning of stained sodium dodecyl sulfate (SDS) gels or quantitation of protein-associated GDP. 5 23 F. W. Studier and B. A. Moffatt, J. Mol. Biol. 189, 113 (1986). 24 O. Weiss, J, Holden, C. Rulka, and R. A. Kahn, J. Biol. Chem. 264, 21066 (1989). 25 R. J. Duronio, E. Jackson-Machelski, R. O. Heuckeroth, P. O. Olins, C. S. Devine, W. Yonemoto, L. W. Slice, S. S. Taylor, and J. I. Gordon, Proc. Natl. Acad, Sci. USA 87, 1506 (1990). 26 j. K. Northup, M. D. Smigel, and A. G. Gilman, J. Biol. Chem. 257, 11416 (1982).
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Protein Expression High-level expression of several ARF and ARF-related proteins has been achieved by using the T7 polymerase/promoter system described by Studier et a/. 23,27 The construction of the ARF expression plasmid, pOW12, has been described previously.24 Briefly, this entails insertion of the coding region of the gene into the pET3C vector~3,27 such that the initiating methionine is part of an NdeI restriction site that is located at the optimal distance from a T7 promoter to direct maximal expression. BL2 I(DE3) cells27 contain the T7 polymerase structural gene under control of the lacUV5 promoter and is thus induced by isopropyl-fl-othiogalactopyranoside (IPTG). BL21 (DE3) cells transformed with pOW 12 are grown in LB medium containing 100/tg/ml ampicillin at 37 °. When the A~0o~- 1.0, IPTG is added to a final concentration of 1 mM. Ninety minutes later, the cells are harvested by centrifugation at 4000 g for 15 min at 4 ° and may be stored at - 8 0 °.
Purification Purification of the recombinant protein is accomplished in two steps, batch elution from DEAE-Sephacel and a gel-filtration column. This protocol routinely results in a product that is at least 85% pure and has been used successfully for recombinant human A R F I I ~ and ARF4p, II drosophila ARL lp, 13and wild-type and eight point mutants of yeast ARFI p.2S 1. The cell pellet from a l-liter culture is resuspended in 20 ml of 50 m M Tris-HC 1, pH 8.0, 40 m M ethylenediaminetetraacetic acid (EDTA), 25% (w/v) sucrose, I mg/ml lysozyme and incubated at room temperature for 15 min. The cells are lysed by the addition of 8 ml of 0.2% (w/v) Triton X-100, 50 mMTris-HC1, pH 8.0, 100 m M MgC12. After l0 rain at 4 °, t h e suspension is clarified by centrifugation at 100,000-g for 60 rain at 4*. 2. The supernatant is loaded onto a 50-ml DEAE-Sephacel column, previously equilibrated in three column volumes of 20 m_MTris-HC1, pH 7.4, 1 m M EDTA, 1 m M dithiothreitol, 50 m M NaC1. The flow through and a wash of one column volume are collected and concentrated to 4 ml by ultrafiltration using an Amicon (Danvers, MA) YM 10 membrane. 3. The concentrate is applied to an Ultrogel AcA 54 column (200 ml, 40 X 2.5 cm; IBF Biotechnics, Columbia, MD) that has been equilibrated with 20 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, 1 m M EDTA, 100 m M NaCl, 1 m M dithiothreitol 27 A. H. Rosenberg, B. N. Lade, D. Chui, S. W. Lin, J. J. Dunn, and F. W. Studier, Gene 56, 125 (1987). 28 R. A. Kahn, J. Clark, and C. Rulka, unpublished observations, 1990.
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(DTT), and 2 m M MgC12. The column is developed in the same buffer at a flow rate of 20 ml/hr and 2.5-ml fractions are collected. The Coomassie blue-stained polyacrylamide gels of protein from each step of the purification are shown in Fig 1. The fractions containing pure ARF are pooled and concentrated by ultrafiltration to I mg/ml and stored at - 80 °.
GTP-BindingAssay ADP-ribosylation factor is easily detected by nucleotide binding and this is the method used in our laboratory for monitoring the elution of recombinant ARF from chromatographic columns during purification. As previously described,5 nucleotide exchange on ARF requires phospholipid and detergent and is most efficient at low concentrations of Mg2+. Hence, the binding cocktail contains 25 m M HEPES, pH 7.4-8.0, with
68
---,"
4 3 ---,"
29
"-
18-"-'-
1
2
3
4
5
FIG. 1. Purification of mARFlp from bacteria. Coomassie blue stained-SDS polyacrylamide gel fractionation of proteins from each step of the purification of mARFlp from BL21 (DE3) cells transfected with pOW12. Lane l, total cell extract from cells induced with IPTG; lane 2, pooled DEAE-Sephacel fractions; lane 3, pooled Ultrogel AcA 54 fractions (purified recombinant mARFlp); lane 4, pooled Ultrogel AcA 54 fractions from bacteria transfected with expression vectors for mARF1 and N-myristoyltransferase; lane 5, ARF purified from bovine brain as previously described.4 Molecular weight markers ( X 10-s) on left-hand side.
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100 mMNaC1, 1 m M EDTA, 0.5 mMMgCI2, 1 m M D T T , 3 m M L-c~dimyristoylphosphatidylcholine(DMPC), 0.1% (w/v) sodium cholate, and 1 a M [ot-32p]GDP (I 0,000 cpm/pmol), [y-32p]GTP (10,000 cpm/pmol), or [35S]GTPyS (10,000 cpm/pmol).4,5 The DMPC is freshly prepared by sonication of a 30 m M solution in 20 m M HEPES, 1 m M EDTA, and 2 m M M g C I 2 immediately prior to performing the binding assay. The turbid suspension is sonicated until it becomes translucent and then mainmined at 30 ° until diluted into the binding cocktail. The protein is incubated in the binding cocktail for 1 hr at 30 ° in a total volume of 50/~1 and exchange is stopped by the addition of ice-cold TNMD buffer: 2 ml of Tris, pH 7.4, 100 mMNaC1, 10 mMMgC12, and 1 m M D T T . The bound and free nucleotides are separated by filtration through 25-mm BA85 nitrocellulose filters (Schleicher & Schuell, Keene, NH). The filters are washed six times with 2 ml ice-cold TNMD. ADP-ribosylation factor-nucleotide complex is retained and can be quantified by scintillation spectroscopy. Binding typically increases linearly for the first 30 min and reaches a plateau within 1 to 3 hr. Binding stoichiometries tend to be poor, especially for triphosphates and triphosphate analogs, as the protein contains 1 mol bound GDP/mol protein. As GDP has at least a 100-fold greater affinity for the protein than the triphosphates the stoichiometry of GTP binding is no more than 0.1. Hence, ARF is not easily quantified by this assay.
Quantitation Typically, the ARF prepared by the protocol above is 85% pure (Fig. 1) which can be assessed by Coomassie blue staining of protein fractionated on polyacrylamide gels. As active ARF is purified as a 1 : 1 complex with GDP, ~ the concentration of active ARF can also be assessed by determining the amount of nucleotide released after heat denaturation of a given amount of the purified protein. 5,24
Comments 1. The time of induction can be varied. With some proteins, accumulation continues beyond 90 min and yields are improved with longer (3-4 hr) inductions. 2. Batch elution from DEAE-Sephacel is the most variable step. Elution with higher concentrations of NaCI increases the yield of ARF but generally results in a less pure preparation after gel filtration. A significant fraction of ARF (as much as 50%) is also found in the 100,000 g pellet. This has been purified in the presence of 7 M urea and results in a nucleotide-free preparation of ARF that is much less stable24 than ARF purified by the protocol presented in this chapter.
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3. This approach has provided our laboratory with purified recombinant ARF proteins from several sources. It has also been modified to obtain myristoylated recombinant proteins. In this case, the bacteria are cotransfected with the plasmid containing the ARF coding region as well as pBB131, 25 a plasmid containing the yeast N-myristoyltransferase gene N M T 1 under the same T7 promoter,25 and a marker for kanamycin resistance to allow selection for both plasmids. The BL21(DE3) cells are then grown in LB medium containing 50/~g/ml kanamycin and 100/zg/ml ampicillin. On induction with IPTG we also add 200 # M myristic acid [125 m M stock in 50% (v/v) methanol is freshly prepared]. Purification, GTP-binding determination, and quantitation are all performed as for the nonmyristoylated protein. Using this approach, approximately 10-60% of the ARF is myristoylated as determined by sequencing of the amino terminus of the purified proteins.28 The myristoylated protein cannot be separated from the unmodified protein using ion-exchange, gel-filtration, or hydrophobic interaction chromatography, including phenyl-Sepharose or heptylamine-Sepharose.28 As is true for the nonmyristoylated recombinant protein, the myristoylated recombinant protein is indistinguishable from ARF purified from bovine brain in terms of nucleotide binding kinetics and activity in the cholera toxin-catalyzed reaction. 2s
[35] By P E T E R
Ypt Proteins in Yeast
WAGNER, LUDGER HENGST,
and
DIETER GALLWITZ
Introduction The ras superfamily of genes encodes structurally related, guanine nucleotide-binding proteins of similar size (around 200 amino acid residues) and biochemical properties. 1-3 From work with yeast mutants, it is evident that members of the Ypt subfamily of proteins,4 including Ypt 1
t H. R. Bourne, D. A. Sanders, and F. McCormick, Nature (London) 348, 125 (1990). 2 H. R. Bourne, D. A. Sanders, and F. McCormick, Nature (London) 349, 117 (1991). 3 A. Hall, Science 249, 635 (1990). 4 D. Gallwitz, H. Haubruck, C. Molenaar, R. Pran__ge,M. Puzieha, H. D. Sehmitt, C. Vorgias, and P. Wagner, in "The Guanine-Nucleotide Binding Proteins" (L. Bosch, B. Kraal, and A. Parmeggiani, eds.), NATO ASI Ser. A, Vol. 165, p. 257. Plenum, New York, 1989.
METHODS IN ENZYMOLOGY, VOL. 219
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