Preface GTPases are now recognized as essential components for protein traffic between all compartments of the cell. This includes vesicular traffic through the exocytic and endocytic pathways, where GTPases play key roles in the assembly of vesicle coats (budding), in vesicle targeting and in fusion, as well as in protein traffic in and out of the nucleus. GTPases involved in transport include the Rab and ARF families, Sarl, Ran, dynamin, and heterotrimeric G proteins. In addition to GTPase, a number of associated accessory factors are critical for function. These include posttranslational modifying enzymes (such as prenyl transferases and myristyl transferases), factors that affect guanine nucleotide binding [guanine nucleotide dissocia- tion inhibitors (GDIs) and guanine nucleotide exchange factors (GEFs)], and factors that stimulate guanine nucleotide hydrolysis [GTPase-activating proteins (GAPs)]. To understand the function of GTPases and their cognate factors, a wealth of in vitro biochemical and in vivo molecular genetic approaches are currently being applied to individual proteins. Given the diverse spectrum of compartments regulated by individual GTPases, techniques developed for one particular member of a family are often applicable to other members. In a broader sense, many of the techniques developed for a particular gene family are also frequently applicable to other gene families given the exceptional structural configuration of GTPases. The purpose of this volume is to bring together the latest technologies in the study of GTPase function involved in protein trafficking. It provides concise descriptions of the recent methodological innovations that allow both the novice and experienced investigator to explore the function of these proteins in detail. We are extremely grateful to the many investigators who have generously contributed their time and expertise to bring this wealth of technical experience to one volume. It should provide a valuable resource to address the many issues confronting our understanding of the role of these GTPases in the biology of cell. W. E. BALCH CHANNING J. DER ALAN HALL xiii Contributors to Volume 257 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. KIRILL ALEXANDROV (27), Cell Biology Pro- gram, European Molecular Biology Labo- ratory, 69012 Heidelberg, Germany SCOTt A. ARMSTRONG (5), Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235 WILLIAM E. BALCH (1, 7, 10, 20, 21), Depart- ments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, Califor- nia 92037 CHARLES BARLOWE (13), Department of Bio- chemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 F. RALF BISCHOFF (17), Division for Molecu- lar Biology of Mitosis, German Cancer Re- search Center, D-69009 Heidelberg, Germany WILLIAM H. BRONDYK (14, 23), Promega Cor- poration, Madison, Wisconsin 53771 MICHAEL S. BROWN (5), Department of Mo- lecular Genetics, University of Texas South- western Medical Center, Dallas, Texas 75235 H. ALEX BROWN (33), Department of Phar- macology, Southwestern Medical Center, University of Texas, Dallas, Texas 75235 CECILIA BUCCI (2, 19), Dipartimento di Bio- logia e Patologia Cellulare e Molecolare "L. Califano, '" 80131 Napoli, Italy HERMAN BUJARD (24), Zentrum far Moleku- late Biologic der Universiti~t Heidelberg, D-69120 Heidelberg, Germany JANET L. BURTON (12), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 ix HANNA DAMKE (24), Department of CeU Biol- ogy, The Scripps Research Institute, La Jolla, California 92037 CHRISTIANE DASCHER (20, 21), Department of Cell Biology, The Scripps Research Insti- tute, La Jolla, California 92037 PIETRO DE CAMILLI (12), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 A. BARBARA DIRAC-SVEJSTRUP (3), Depart- ment of Biochemistry, Stanford University School of Medicine, Stanford, California 943O5 CARLOS G. DOTTI (32), Cell Biology Pro- gram, European Molecular Biology Labo- ratory, D-69117 Heidelberg, Germany PAUL DUPREE (32), Department of Plant Sci- ences, Cambridge University, Cambridge CB2 3HA, United Kingdom MARILYN GIST FARQUHAR (29), Division of Cellular and Molecular Medicine, Univer- sity of California, San Diego, La Jolla, Cali- fornia 92093 SUSAN FERRO-NovICK (4), Department of Cell Biology, Howard Hughes Medical In- stitute, Yale University School of Medicine, New Haven, Connecticut 06510 SABINE FREUNDIAEB (24), Zentrum far Mo- lekulare Biologie der Universitiit Heidel- berg, D-69120 Heidelberg, Germany DIETER GALLWlTZ (15), Department of Mo- lecular Genetics, Max-Planck Institute for Biophysical Chemistry, D-37018 GOt- tingen, Germany MICHELLE D. GARRE~rr (11, 26), Onyx Phar- maceuticals, Richmond, California 94806 X CONTRIBUTORS TO VOLUME 257 LARRY GERACE (30), Department of Cell Bi- ology, The Scripps Research Institute, La Jolla, California 92037 JOSEPH L. GOLI~STEIN (5), Department of Mo- lecular Genetics, University of Texas South- western Medical Center, Dallas, Texas 75235 MANFRED GOSSEN (24), MCB Barker/Kosh- land ASU, University of California, Berke- ley, California 94720 RONALD W. HOLZ (25), Department of Phar- macology, University of Michigan Medical School, Ann Arbor, Michigan 48109 HISANORI HORIUCHI (2, 27), CelIBiology Pro- gram, European Molecular Biology Labo- ratory, 69012 Heidelberg, Germany Lugs A. HUBER (32), Department of Bio- chemistry, University of Geneva, CH-1211 Geneva 4, Switzerland Yu JIANG (4), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 RICHARD A. KAHN (16), Laboratory of Bio- logical Chemistry, Division of Cancer Treatment, National Cancer Institute, Na- tional Institutes of Health, Bethesda, Mary- land 20892 AKIRA KIKUCHI (8), Department of Biochem- istry, Hiroshima University School of Medi- cine, Hiroshima 734, Japan KEITaROU KIMU~ (6), Genetics Engineering Laboratory, National Food Research Insti- tute, Tsukuba 305, Japan IAN G. MaCARA (14, 23), Department of Pa- thology, University of Vermont, Burlington, Vermont 05405 Luis MARTIN-PARRAS (22), Cell Biology Pro- gram, European Molecular Biology Labo- ratory, D-69117 Heidelberg, Germany J. MICHAEL MCCAFFERY (29), Division of Cellular and Molecular Medicine, Univer- sity of California, San Diego, LaJolla, Cali- fornia 92093 FRAUKE MELCHIOR (30), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 CAROL MURPHY (34), Cell Biology Program, European Molecular Biology Laboratory, D-69012 Heidelberg, Germany HIROYUKI NAKANISHI (8), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan AKImRO NAga~YO (6), Department of Biologi- cal Sciences, Graduate School of Science, University of Tokyo, Tokyo 113, Japan PETER J. NOVICK (1l, 26), Department of Cell Biology, Yale University School of Medi- cine, New Haven, Connecticut 06510 CLAUDE NUOFFER (1, 10), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 TOSHmIKO OKA (6), Department of Organic Chemistry and Biochemistry, Institute of Scientific and Industrial Research, Osaka University, Osaka 567, Japan FRANK PETER (1, 10), Department of Cell Bi- ology, The Scripps Research Institute, La Jolla, California 92037 SUZANNE R. PFEFVER (3, 28), Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 HERWlG PONSa~NGL (17), Division for Molec- ular Biology of Mitosis, German Cancer Research Center, D-69009 Heidelberg, Germany PAUL A. RANDAZZO (16), Laboratory of Bio- logical Chemistry, Division of Cancer Treatment, National Cancer Institute, Na- tional Institutes of Health, Bethesda, Mary- land 20892 MARKUS A. RmDERER (3), Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 DENISE M. ROBERTS (11), Department of Cell Biology, Yale University School of Medi- cine, New Haven, Connecticut 06510 GUENDALINA ROSSI (4), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 TONY ROWE (7), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 CONTRIBUTORS TO VOLUME 257 xi TAKUYA SASAKI (9), Department of Molecu- lar Biology and Biochemistry, Osaka Uni- versity Medical School, Suita 565, Japan ISABELLE SCHALK (10), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 RANDY SCHEK/vIAN (13, 18), Departments of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720 SANDRA L. SCHMID (24), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 MIGUEL C. SEABRA (5), Department of Molec- ular Genetics, University of Texas South- western Medical Center, Dallas, Texas 75235 RUTH A. SENTER (25), Department of Phar- macology, University of Michigan Medical School, Ann Arbor, Michigan 48109 ALLAN D. SHAPIRO (28), Department of Bio- chemistry, Stanford University School of Medicine, Stanford, California 94305 HIROMICHI SHIRATAKI (31), Department of Cell Biology, National Institute for Physio- logical Sciences, Okazaki 444, Japan THIERRY SOLDATI (3, 28), Department of Bio- chemistry, Stanford University School of Medicine, Stanford, California 94305 HARALD STENMARK (19), Cell Biology Pro- gram, European Molecular Biology Labo- ratory, D-69012 Heidelberg, Germany PAUL C. STERNWEIS (33), Department of Pharmacology, University of Texas South- western Medical Center, Dallas, Texas 75235 DEBORAH J. SWEET (30), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 YOSHIMI TAKAI (8, 9, 31), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School and Department of Cell Physiology, National Institute for Physiological Sciences, Suita, Osaka 565, Japan LAUREL THOMAS (21), Vollum Institute, Ore- gon Health Sciences University, Portland, Oregon 97201 GARY THOMAS (21), VoUum Institute, Oregon Health Sciences University, Portland, Ore- gon 97201 ELLEN J. TISDALE (20), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 MICHAEL D. UHLER (25), Department of Bio- logical Chemistry and The Mental Health Research Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109 OLIVER ULLRICH (2, 27), Cell Biology Pro- gram, European Molecular Biology Labo- ratory, 69012 Heidelberg, Germany JUDY K. VANSLYKE (21), Vollum Institute, Oregon Health Sciences University, Port- land, Oregon 97201 PETRA VOLLMER (15), Department of Molecu- lar Genetics, Max Planck Institute for Bio- physical Chemistry, D-37018 Gottingen, Germany OFRA WEISS (16), Department of Endocrinol- ogy and Metabolism, Hadassah University Hospital, Jerusalem 91120, Israel THOMAS YEUNG (18), Division of Biochemis- try and Molecular Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720 TOHRU YOSHIHISA (18), Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan MARINO ZERIAL (2, 19, 22, 27, 34), Cell Biol- ogy Program, European Molecular Biology Laboratory, D-69012 Heidelberg, Germany [ 1] PURIFICATION OF His6-Rabl 3 [11 Purification of His6-Tagged Rabl Proteins Using Bacterial and Insect Cell Expression Systems By CLAUDE NUOFFER, FRANK PETER, and WILLIAM E. BALCH Introduction The members of the Rab/YPT/SEC4 family of Ras-like GTPases are likely to function as molecular switches' in regulating the assembly and/or disassembly of protein complexes that mediate the vectorial movement of transport vesicles between distinct subcellular compartments. We have established that the Rabl proteins play an essential role in traffic through the early secretory pathway in mammalian cells by showing that selected RablA and RablB mutants with altered guanine nucleotide-binding prop- erties act as potent trans dominant inhibitors of transport between the endoplasmic reticulum (ER) and the Golgi complex both in vivo t and in vitro. 2'3 This chapter describes the isolation of recombinant wild-type or mutant forms of Rabl via expression in Escherichia coli and Spodoptera frugiperda (Sf9) insect cells. Although the bacterial expression system is more convenient from a technical point of view, the utility of Rabl proteins prepared from E. coli is limited by the fact that these invariably lack the COOH-terminal geranylgeranyl (GG) groups that are essential for normal Rabl function. 2 In contrast, the eukaryotic expression system allows the purification of membrane-associated, isoprenylated forms of the proteins (RablGG). 4 Both expression systems require the purification of relatively minor pools of functional protein. In the case of E. coli, this is due to the strong tendency of Rabl proteins to form inclusion bodies. To obtain active forms of the proteins we focus on the purification of the soluble pool, which represents no more than 1-10% of the total production. In Sf9 cells, the yields of isoprenylated Rabl proteins are low as <5% of the expressed protein is posttranslationally processed and incorporated into host cell membranes. To overcome these difficulties, 1 E. J. Tisdale, J. R. Bourne, R. Khosravi-Far, C. J. Der, and W. E. Balch, J. Cell BioL 119, 749 (1992). 2 C. Nuoffer, H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, J. Cell Biol. 125, 225 (1994). 3 S. Pind, S. N. Pind, C. Nouffer, J. M. McCafffey, H. Plutner, H. W. Davidson, M. G. Farquhar, and W. E. Balch, J. Cell Biol. 125, 239 (1994). 4 F. Peter, C. Nuoffer, S. N. Pind, and W. E. Balch, J. Cell Biol. 126, 1393 (1994). Copyright © 1995 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 257 All rights of reproduction in any form reserved. 4 EXPRESSION, PURIFICATION, AND MODIFICATION [ 1] we take advantage of N-terminal His6 tags that allow us to use metal chelate chromatography as a rapid and efficient purification step. 5 These N-terminal His6 modifications do not interfere with the function of wild-type and mutant Rabl proteins in transport through the early secretory pathway. 2 Methods Purification of His6-Rab i from Escherichia coli His6-Rabl proteins are produced in E. coli using the T7 RNA polymer- ase-dependent expression system developed by Studier et al. 6 Briefly, the cDNA is placed under control of a T7 promoter and the resulting expression vector is introduced into E. coli strain BL21(DE3), which contains the T7 RNA polymerase gene under control of the lacZ promoter. Exposure of the cells to isopropyl-/3-thiogalactopyranoside (IPTG) induces T7 RNA polymerase production and triggers expression of the cDNA. Procedures Buffers Lysis buffer: 50 mM Tris-HC1, pH 8,1 mM EDTA, 10 mM 2-mercapto- ethanol NTA buffer: 50 mM MES-NaOH, pH 6, 0.3 M NaCI, 1 mM MgCI2, 50/zM EGTA, 10 mM 2-mercaptoethanol 25/125:25 mM HEPES-KOH, pH 7.2, 125 mM potassium acetate Construction of Expression Vectors An expression vector for the production of Rab proteins with a N-terminal His6 tag was first constructed using the Rab3A cDNA and plasmid pETlld (Novagen) as follows: A NcoI-NdeI linker encoding an initiator Met followed by six consecutive His residues was ligated along with the Rab3a cDNA excised from pET3a-Rab3A as a NdeI-BamHI fragment into the NcoI and BamHI sites of pETlld. 2 Constructs directing the expression of wild-type and mutant His6-Rabl proteins were obtained through excision of the Rab3a sequence with NdeI and BamHI and inser- tion of the corresponding Rabl fragments isolated from pET3a-Rabl plasmids, m 5 E. Hochuli, W. Bannwarth, H. DObeli, R. Gentz, and D. StOber, Bio Technology 6,1321 (1988). 6 F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, this series, Vol. 185, p. 60. [ 1] PURIFICATION OF His6-Rabl 5 Expression The pETlld-His6-Rabl plasmids are introduced into competent BL21(DE3) cells and transformants are selected on LB-agar plates con- taining 100/xg/ml ampicillin overnight at 37 °. A single colony is transferred into LB supplemented with 100 tzg/ml ampicillin, and the preculture is grown to saturation overnight at 37 °. The culture is diluted 1 : 50 into fresh medium and grown at 28! to OD60o of 0.6-1.0 with good aeration. Expres- sion is induced by the addition of IPTG to a final concentration of 0.4 mM and incubation is continued for 2-4 hr. The cultures are chilled on ice, the cells are harvested by centrifugation, washed, and the cell pellets are frozen in liquid N2 and stored at -80 °. Note: The expression protocol just described results in levels of soluble protein that vary considerably between different wild-type and mutant forms of Rabl. In some cases, induction for 6-16 hr in the presence of 0.01-0.1 mM IPTG may result in higher yields of soluble protein. In general, mutant forms of Rabl tend to be less soluble compared to the wild-type proteins. This is most evident in the case of the RablA/B(N124/121I) mutants, 3 which are extremely insoluble and remain prone to precipitation throughout the purification process and cannot be kept in solution at con- centrations >0.2 mg/ml. Purification Preparation of L ysis Supernatant All subsequent manipulations are performed at 4 ° unless otherwise stated. The pellets are thawed and resuspended in 10 vol lysis buffer supple- mented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5/xg/ml leu- peptin, and 1/xM pepstatin A. Lysozyme is added to a final concentration of 0.4 mg/ml and the suspension is incubated for 30 rain at 4 ° with gentle agitation. After lysis of the cells through two rounds of freezing in liquid N2 followed by thawing at 32 ° with constant agitation, the lysate is adjusted to 0.3 M NaCI, 10 mM MgCI2, and 0.2% deoxycholate. The viscosity is reduced by incubation in the presence of 40 gg/ml DNase I for 30 min at 4 ° with gentle agitation, and the lysate is clarified by centrifugation at 22,000g (13,500 rpm in a Beckman JA-20 rotor) for 30 min. The resulting supernatant serves as a source to purify the soluble His6-Rabl fraction by metal chelate affinity chromatography and gel filtration chromatography as described below. Note: The inclusion of 10/xM GDP in the lysis buffer and throughout the remainder of the purification process may slightly increase the stability 6 EXPRESSION, PURIFICATION. AND MODIFICATION [1] of Rabl mutants with low affinities for guanine nucleotides such as the RablA/B(N124/121I) mutants. 3 Ni2+-NTA-Agarose Chromatography The supernatant is applied to a column (0.5-5 ml bed volume) of Ni 2+- saturated nitrilotriacetic acid (NTA)-agarose (Qiagen) equilibrated with lysis buffer containing 0.3 M NaC1 and 10 mM MgCI2 (flow rate: 1 ml/ min). The column is washed with 10 vol of equilibration buffer, 10 vol of NTA-buffer, and 10 vol of NTA-buffer supplemented with 25 mM imidaz- ole. The column is eluted with NTA-buffer containing 250 mM imidazole, and fractions containing His6-Rabl are identified by analyzing aliquots by SDS-PAGE and Coomassie blue staining. Fractions containing His6-Rabl are pooled, and the proteins are further purified by gel filtration chromatog- raphy. Note: To minimize the nonspecific adsorption of proteins to the NTA- agarose resin, it is essential to adjust the bed volume of the NTA-agarose column depending on the amount of His6-Rabl present in the lysate. For the purification of wild-type proteins and mutants with comparable solubility [RablA/B(S25/22N)2], we typically use -1-2 ml of resin for each liter of culture. In the case of the RablA/B(N124/121I) mutants, better results are obtained with -5-10x smaller columns. S-I O0 Gel-Filtration Chromatography The pooled fractions are applied to a 75 x 2.5-cm column (flow rate: -0.5 ml/min) of Sephacryl S-100 (Pharmacia LKB) equilibrated with 25/125 supplemented with 1 mM MgCI2 and 1 mM sodium mercaptoethanesulfonic acid. Fractions containing His6-Rabl are identified by analyzing aliquots by SDS-PAGE and Coomassie blue staining. The proteins elute with an apparent molecular mass of -24-26 kDa. Peak fractions are pooled and concentrated by ultrafiltration using Centricon concentrators (Amicon Danvers, MA). Aliquots are frozen in liquid N2 and stored at -80 °. Note: In the case of the wild-type proteins and the RablA/B(S25/22N) mutants, -1-2.5 mg of >95% pure His6-Rabl can be recovered per liter of culture. The yields are typically -10-20x lower for the RablA/B(N124/ 1211) mutants. Comment Recombinant proteins isolated from E. coli have been used to determine the guanine nucleotide-binding properties of various Rabl mutants. 2,3 Moreover, we have shown that the RablA/B(N124/121I) mutants do not require posttranslational processing to perturb transport between the endo- [ 1] PURIFICATION OF His6-Rabl 7 plasmic reticulum and the Golgi complex in vivo and in 12itro. 1'2 In contrast, the COOH-terminal geranylgeranyl modifications are essential for wild- type Rabl function and the inhibitory activity of the RablA/B(S25/22N) mutants. 2 It is possible, however, to convert a fraction of these proteins into the biologically active form in vitro by incubation in the presence of exogenous geranylgeranyl pyrophosphate and rat liver cytosol as a source of rab geranylgeranyltransferase, 2 even though the efficiency of this reaction is relatively low. Purification of His6-RablGG from Sf9 Membranes His6-Rabl proteins are produced in Sf9 cells following infection of the cells with high titer stocks of recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) which direct the expression of the cloned cDNAs under control of the viral polyhedrin promotor. 7 Procedures Buffers Lysis buffer: 50 mM HEPES-KOH, pH 7.2, 1 mM MgClz Extraction buffer: Lysis buffer supplemented with 0.15 M NaC1 and 0.6% 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfo- nate (CHAPS) Mono Q buffer: 25 mM Tris-HC1, pH 7.5, 1 mM MgCI2, 0.6% CHAPS Generation of Recombinant Virus Recombinant virus stocks were prepared using the MaxBac baculovirus expression vector system (Invitrogen). cDNA fragments with flanking NheI sites were amplified by polymerase chain reaction from the respective pET-Rabl constructs (see above) using appropriate 5'- and 3'-oligonucleo- tide primers according to standard procedures. The products were sub- cloned, verified by DNA sequencing, and introduced into the NheI site of the baculovirus transfer vector pBlueBac. Constructs containing a single insert in the appropriate orientation were selected by restriction analysis, and the pBlueBac-His6-Rabl plasmids were cotransfected along with linear AcMNPV DNA into Sf9 cells. Viral recombinants were identified, purified, amplified, and titered according to the instructions of the manufacturer. High titer stocks ( 1-2 × 108 plaque-forming units/ml) are stored in ali- quots at 4 ° in the dark. 7 M. D. Summers and G. E. Smith, Tex., Agric. Exp. Stn. [Bull.] 1555 (1987). 8 EXPRESSION, PURIFICATION, AND MODIFICATION [1 ] Expression Sf9 cells are grown in Ex-Cel1400 (JRH Bioscience) supplemented with 5% fetal bovine serum to a density of -1.5-2.5 × 106 cells/ml in spinner flasks that are maintained at 26-27 ° . The cells are infected with recombinant virus at a multiplicity of infection of 5-10 and incubation is continued for 72 hr. The cells are harvested and washed with phosphate-buffered saline, and cell pellets are resuspended in 2 vol of lysis buffer, frozen in liquid N2, and stored at -80 ° . Preparation of Membrane Fraction and Membrane Extraction All subsequent manipulations are performed at 4 ° unless otherwise stated. The cell suspension is thawed and diluted with 1 vol of lysis buffer supplemented with 0.3 M NaC1, 1 mM PMSF, 0.5/~g/ml leupeptin, and 1 /zM pepstatin. Lysis is accomplished by using a N2 cavitation bomb (25 min, 500 psi). The homogenate is centrifuged for 5 min at 900g to remove cell debris and nuclei, and membranes are pelleted from the supernatant by centrifugation at 100,000g for 1 hr (40,000 rpm in a Beckman Ti60 rotor). The membranes are resuspended in 10 vol of lysis buffer supplemented with 0.15 M NaCI and the protease inhibitor cocktail using a Dounce homogenizer and centrifuged again as described earlier. The washed mem- brane pellets are resuspended in 5 vol of extraction buffer supplemented with the protease inhibitor cocktail, and the extracts are clarified by centrifu- gation as described previously. The supernatant is used to purify His6- RablGG by metal chelate chromatography followed by anion-exchange chromatography on a Mono Q FPLC (fast protein liquid chromatography) column as described below. Note: Complete lysis of the cells prior to the high-speed centrifugation is essential to minimize contamination of isoprenylated RablGG with soluble cytosolic Rabl lacking the COOH-terminal geranylgeranyl groups. The nonprocessed pool can be purified from the cytosolic fraction essentially as described earlier for the purification of His6-Rabl from E. coli lysis super- natants. Purification Ni2 +-NTA-Agarose Chromatography Sf9 membrane extracts are processed on Ni2+-NTA-agarose columns as described for E. coli lysates, except that all buffers are supplemented with 0.6% CHAPS. [...]... from Sf9 Cells Construction and Selection of Rab5-Containing Baculovirus A full-length cDNA-encoding canine Rab512 is cloned in the BamHI site downstream of the polyhedrin promoter in the baculovirus transfer vector pVL1393.13 A Rab5 recombinant Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) is constructed by homologous recombinationJ 4 Briefly, 1 /xg of linear A c M N... A BARBARA D I R A C - S V E J S T R U P , and SUZANNER PFEFFER Introduction This chapter describes the purification of canine Rab9 after expression in Escherichia coli, and the small- scale and preparative-scale isoprenylation of Rab9 in vitro Escherichia coli-expressed Rab proteins are valuable reagents in analyzing the biochemical properties, structural features, and functional activities of individual... t B) and an accessory subunit (also called c o m p o n e n t A) 6-1° The catalytic c o m p o n e n t is a heterodimer composed of a a n d / 3 subunits 6,7 The accessory c o m p o n e n t is a single polypeptide that functions as an escort protein This subunit presents substrate to the catalytic component of the enzyme 8,l° In the yeast Saccharomyces cerevisiae, the/3 subunit of the catalytic c o m... FERRO-NOVICK Introduction Members of the Rab GTP-binding protein family are involved in the regulation of different exocytic and endocytic transport processes 1 They are localized to diverse intracellular compartments and participate in various steps of vesicular traffic.1 In yeast, two Rab GTPases, Sec4p and Yptlp, have been shown to play a role on the exocytic pathway 2'3 They are significantly homologous... Simons, and M Zerial, Cell (Cambridge, Mass.) 62, 317 (1990) 9j_p Gorvel,P Chavrier, M Zerial, and J Gruenberg, Cell (Cambridge, Mass.) 64, 915 (1991) l0 C Bucci, R G Parton, I H Mather, H Stunnenberg, K Simons, B Hoflack, and M Zerial, Cell (Cambridge, Mass.) 70, 715 (1992) 11O Ullrich, H Horiuchi, C Bucci, and M Zerial, Nature (London) 368, 157 (1994) [2] PURIFICATIONOF Rab5 PROTEIN 11 Purification... Goldstein, Cell (Cambridge, Mass.) 70, 1049 (1992) 10D A Andres, M C Seabra, M S Brown, S A Armstrong, T E Smeland, F P M Cremers, and J L Goldstein, Cell (Cambridge, Mass.) 73, 1091 (1993) 11M L Mayer, B E Caplin, and M S Marshall, J Biol Chem 267, 20589 (1992) 12B He, P Chen, S.-Y Chen, K L Vancura, S Michaelis, and S Powers, Proc Natl Acad Sci U.S.A 88, 11373 (1991) 13j F Moomaw and P J Casey, J Biol Chem... the coding sequence of lacZ The other primer contains a ClaI site that is downstream from the termination codon The PCR product is then digested with KpnI and ClaI and is ligated into the KpnI and ClaI sites of pBC-KS to yield pSFN172 The fusion product (/3-galactosidase-Bet4p-1) generated by this construction contains the first 20 amino acids of/3-galactosidase followed by the Bet4p sequence (lacking... carboxy-terminal cysteine residues (CC) of Yptlp were replaced with a CXC motif We found that this Yptlp derivative is modified as efficiently as the wild-type protein 7 This indicates that the yeast GGTase II, like its mammalian counterpart, modifies proteins that terminate in a CC and CXC motif To further characterize the yeast enzyme, we have expressed Bet2p/Bet4p and Mrs6p in bacterial cells to reconstitute activity... to dissect G G T a s e II activity further in terms of the substrate specificity of the enzyme and the subunit composi6 M C Seabra, J L Goldstein, T C Sudhof, and M S Brown,J Biol Chem 267,14497 (1992) 7y Jiang, G Rossi, and S Ferro-Novick, Nature (London) 366, 84 (1993) 8 y Jiang and S Ferro-Novick, Proc Natl Acad Sci U.S.A 91, 4377 (1994), 9 M C Seabra, M S Brown, C A Slaughter, T C Sudhof, and J L... the induced E coli lysate and is further purified Amino-terminal sequencing and mass spectrometry have confirmed that the 22-kDa polypeptide represents a truncated form of Rab9 which lacks 22 amino acids at its carboxy terminus 3 Typically, 30-50% of Rab9 is recovered in truncated form, which we refer to as Rab9AC This degradation product is completely resolved from intact Rab9 on Q-Sepharose chromatography . Preface GTPases are now recognized as essential components for protein traffic between all compartments of the cell. This includes vesicular traffic through the exocytic and endocytic pathways,. DIRAC-SVEJSTRUP, and SUZANNE R. PFEFFER Introduction This chapter describes the purification of canine Rab9 after expression in Escherichia coli, and the small- scale and preparative-scale. DIRAC-SVEJSTRUP, and SUZANNE R. PFEFFER Introduction This chapter describes the purification of canine Rab9 after expression in Escherichia coli, and the small- scale and preparative-scale