small gtpases and their regulators, part c

BURTON 12, Department of Cell Biology, Howard Hughes Medical Institute, Yale University School o f Medicine, New Haven, Connecticut 06510 ix HANNA DAMKE 24, Department of CeU Biol-

P r e f a c e 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 A R F 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

C o n t r i b u t o r s to V o l u m e 2 5 7 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 o f 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

L u g s A HUBER (32), Department of Bio-

chemistry, University of Geneva, CH-1211

Geneva 4, Switzerland

Y u 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 K I M U ~ (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

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

R a b l A 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 v i t r o 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,

4 F Peter, C Nuoffer, S N Pind, and W E Balch, J Cell Biol 126, 1393 (1994)

Copyright © 1995 by Academic Press, Inc

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

Construction o f 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 p E T l l d (Novagen) as follows: A N c o I - N d e I linker encoding an

initiator Met followed by six consecutive His residues was ligated along

with the Rab3a cDNA excised from pET3a-Rab3A as a N d e I - B a m H I fragment into the NcoI and B a m H I sites of p E T l l d 2 Constructs directing

the expression of wild-type and mutant His6-Rabl proteins were obtained

through excision of the Rab3a sequence with NdeI and B a m H I and inser-

tion of the corresponding Rabl fragments isolated from p E T 3 a - R a b l 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 p E T l l d - H i s 6 - R a b l 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 m M 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 - 8 0 °

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 m M IPTG may result in higher yields of soluble protein In general, mutant forms of R a b l 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

S D S - P A G E 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 N T A - 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 - 1 0 x 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 m M MgCI2 and 1 mM sodium mercaptoethanesulfonic acid Fractions containing His6-Rabl are identified by analyzing aliquots

by S D S - P A G E and Coomassie blue staining The proteins elute with an apparent molecular mass of - 2 4 - 2 6 kDa Peak fractions are pooled and concentrated by ultrafiltration using Centricon concentrators (Amicon Danvers, MA) Aliquots are frozen in liquid N2 and stored at - 8 0 ° 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 - 1 0 - 2 0 x 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 R a b l 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 R a b l 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 M e m b r a n e s

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

Mono Q buffer: 25 m M Tris-HC1, pH 7.5, 1 m M MgCI2, 0.6% CHAPS

Generation o f 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

p E T - R a b l constructs (see above) using appropriate 5'- and 3'-oligonucleo- tide primers according to standard procedures The products were sub-

cloned, verified by D N A 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 D N A 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 ( J R H 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 - 8 0 °

Preparation o f 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 m M 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-

R a b l G G 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 R a b l G G with soluble cytosolic R a b l 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

121 PURIFICATION OF Rab5 PROTEIN 9

Mono Q Chromatography

Eluates from the NiE÷-NTA-agarose columns are concentrated and dialyzed against 50 vol of Mono Q buffer The sample is diluted to 10 ml, filtered through a 0.22-/zm Durapore membrane (Millipore), and loaded onto an FPLC Mono Q HR5/5 column (Pharmacia) equilibrated with Mono Q buffer (flow rate: 1 ml/min) After washing the column with

20 ml of Mono Q buffer, it is developed with a linear gradient of 0-0.25 M NaCI in Mono Q buffer over 20 min and 1-ml fractions are collected His6-RablGG, which elutes in the range of 50-100 m M NaCI, is identified by analyzing aliquots of the fractions by S D S - P A G E and Coomassie blue staining The fractions are pooled, concentrated, dialyzed against 25/125 containing 0.6% CHAPS, and stored in aliquots

a soluble G D I - R a b l complex in vitro (see [10] in this volume) This complex

has been shown to serve as a functional source of R a b l for vesicular transport between the E R and the Golgi complex in vitro 4

8 C Bordier, J Biol Chem 256, 1604 (1981)

121 PURIFICATION OF Rab5 PROTEIN 9

Mono Q Chromatography

Eluates from the NiE÷-NTA-agarose columns are concentrated and dialyzed against 50 vol of Mono Q buffer The sample is diluted to 10 ml, filtered through a 0.22-/zm Durapore membrane (Millipore), and loaded onto an FPLC Mono Q HR5/5 column (Pharmacia) equilibrated with Mono Q buffer (flow rate: 1 ml/min) After washing the column with

20 ml of Mono Q buffer, it is developed with a linear gradient of 0-0.25 M NaCI in Mono Q buffer over 20 min and 1-ml fractions are collected His6-RablGG, which elutes in the range of 50-100 m M NaCI, is identified by analyzing aliquots of the fractions by S D S - P A G E and Coomassie blue staining The fractions are pooled, concentrated, dialyzed against 25/125 containing 0.6% CHAPS, and stored in aliquots

a soluble G D I - R a b l complex in vitro (see [10] in this volume) This complex

has been shown to serve as a functional source of R a b l for vesicular transport between the E R and the Golgi complex in vitro 4

8 C Bordier, J Biol Chem 256, 1604 (1981)

10 EXPRESSION, PURIFICATION, AND MODIFICATION [2]

geranylgeranyltransferase (Rab GGTase) 1 A l t h o u g h Rab proteins ex-

pressed in Escherichia coli do not undergo this modification, they are active

in guanine nucleotide binding and G T P hydrolysis Factors that modulate

G D P / G T P exchange and G T P hydrolysis have been searched using these proteins 2 However, geranylgeranylation has been shown to be essential

for the function of R a b proteins in v i v o 3 and to interact with one regulatory protein, R a b - G D P dissociation inhibitor ( G D I ) , in vitro R a b - G D I forms

a complex with, and inhibits G D P dissociation from, several Rab pro- teins 4-6 Furthermore, R a b - G D I modulates the m e m b r a n e association of

R a b proteins and is required for their function 7 Therefore, it is important

to obtain posttranslationally modified R a b proteins in order to study the mechanism of their m e m b r a n e association and function

Rab5 is a 25-kDa GTP-binding protein localized to the plasma membrane, clathrin-coated vesicles, and early endosomes, and functions

as a regulatory factor of endocytosis 8-1° As for other R a b proteins, Rab5 is geranylgeranylated at its C terminus 6 and this modification is essential for its function, l° In order to obtain Rab5 in the isoprenylated form, we have made use of a baculovirus expression system This chapter describes a m e t h o d to purify both posttranslationally modified and

unmodified Rab5 from S p o d o p t e r a f r u g i p e r d a (Sf9) insect cells overex-

pressing the protein Purified posttranslationally modified and unmodified Rab5 protein efficiently bind G T P and G D P However, as expected,

R a b - G D I is active only on modified Rab5 W h e n modified Rab5 com- plexed with R a b - G D I is introduced into permeabilized cells, Rab5 is localized to its correct site of function and induces the formation of

enlarged early endosomes as previously observed in vivo, l° indicating

that it is functionally active, ll

1M C Seabra, M S Brown, C A Slaughter, T C Stidhof, and J L Goldstein, Cell (Cambridge, Mass.) 70, 1049 (1992)

E S Burstein and I G Macara, Proc Natl Acad Sci U.S.A 89, 1154 (1992)

3 p Chavrier, J.-P Gorvel, E Steltzer, K Simons, J Gruenberg, and M Zerial, Nature (London) 353, 769 (1991)

4 T Sasaki, A Kikuchi, S Araki, Y Hata, M Isomura, S Kuroda, and Y Takai, J Biol Chem 265, 2333 (1990)

5 S Araki, K Kaibuchi, T Sasaki, Y Hata, and Y Takai, Mol Cell, Biol 11, 1438 (1991)

60 Ullrich, H Stenmark, K Alexandrov, L A Huber, K Kaibuchi, T Sasaki, Y Takai,

and M Zerial, J Biol Chem 268, 18143 (1993)

7 M D Garrett, J E Zahner, C M Cheney, and P J Novick, EMBO J 13, 1718 (1994)

8 p Chavrier, R G Parton, H P Hauri, K Simons, and M Zerial, Cell (Cambridge, Mass.)

62, 317 (1990)

9 j_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)

11 O Ullrich, H Horiuchi, C Bucci, and M Zerial, Nature (London) 368, 157 (1994)

[2] PURIFICATION OF Rab5 PROTEIN 11

P u r i f i c a t i o n of P o s t t r a n s l a t i o n a l l y Modified a n d U n m o d i f i e d R a b 5

f r o m 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 p r o m o t e r in the baculovirus transfer vector pVL1393.13 A Rab5 recombinant Autographa californica multiple

nucleocapsid nuclear polyhedrosis virus ( A c M N P V ) is constructed by ho- mologous r e c o m b i n a t i o n J 4 Briefly, 1 /xg of linear A c M N P V D N A 15 (In- vitrogen) is mixed in a polypropylene tube with 5 tzg of the transfer vector containing the c D N A encoding Rab5 in 120/xl of a buffer [20 m M N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid ( H E P E S ) , p H 7.4, and

150 m M NaC1] In a separate tube, 60/zl of the transfection reagent D O T A P (Boehringer-Mannheim) is added to 60 t~l of the same buffer Both solutions are mixed and incubated at r o o m t e m p e r a t u r e for 15 min T h r e e milliliters

of serum-free Grace's medium ( G I B C O , G r a n d Island, NY) is then added

to the transfection tube Sf9 cells (2.0 × 106 cells), seeded in a 25-cm 2 flask

1 hr before to be allowed to attach to the substratum, are washed twice with the s e r u m 4 r e e medium and then the transfection solution is added

A f t e r 7 hr, 3 ml of Grace's medium supplemented with 20% heat-inactivated fetal calf serum (FCS) is added and the cells are further incubated at

27 ° A f t e r a week the medium is collected and used at different dilutions (10-1-10 -6) to infect Sf9 cells plated 1 hr before at a density of 106/25-cm 2 flask After 1 hr of infection, a plaque assay is p e r f o r m e d as previously described 14 and cells are left at 27 ° After 6 - 8 days, plaques containing putative recombinant virus are selected T h e virus is eluted in the medium and is used for a n o t h e r plaque purification assay R e c o m b i n a n t plaques are identified for the absence of occlusions that are normally f o r m e d on expression of the polyhedrin protein

Expression of Rab5 in Sf9 Cells

Sf9 cells are grown in 165-cm 2 tissue culture flasks (Greiner) in Grace's

m e d i u m supplemented with 10% (v/v) heat-inactivated FCS, 100 U/ml penicillin, and 100/zg/ml streptomycin at 27 ° A virus stock is p r e p a r e d by infecting Sf9 cells with the recombinant virus On the 5th day after infection, the medium is collected and centrifuged at 1000g for 10 rain at 4 ° to r e m o v e

lz p Chavrier, M Vingron, C Sander, K Simons, and M Zerial, MoL Cell Biol 10, 6578 (1990)

13 V A Luckow, in "Recombinant DNA Technology and Applications" (A Prokop, R K

Bajpai, and C S Ho, eds.), p 97 McGraw-Hill, New York, 1991

14 M D Summers and G E Smith, Tex., Agric Exp Stn [Bull.] 1555 (1987)

15 p, A Kitts, M D Ayres, and R D Possee, Nucleic Acids/Res 18, 5667 (1991)

12 EXPRESSION, PURIFICATION, AND MODIFICATION I21 floating cells The supernatant containing the virus is stored at 4 ° as a virus stock For producing Rab5 protein, subconfluent Sf9 cells grown on three 24.5 × 24.5-cm tissue culture plates (Nunc) are infected with 7.5 ml of the virus stock per plate in 75 ml of Grace's media supplemented with 10% (v/v) heat-inactivated FCS, 100 U/ml penicillin, and 100/~g/ml streptomycin and are incubated for 3 days at 27 ° The cells are harvested and pelleted

by centrifugation at 1000g for 10 min at 4 ° After one wash with 50 ml of phosphate-buffered saline, cells are centrifuged again and the pellet (3 ml)

is stored at - 8 0 ° until use Subsequently, the cell pellet is fractionated into a high-speed pellet (membrane fraction) and a supernatant (cytosol fraction) The posttranslationally modified Rab5 is purified from the mem- brane fraction, whereas the cytosol fraction contains large amount of un- modified Rab5

Preparation of Cytosol and Membrane Fractions from Sf9 Cells

The pellet of Rab5-expressing Sf9 cells is resuspended in 20 ml of ice- cold buffer A [20 mM H E P E S / K O H , pH 7.2, 2 m M ethylene glycol bis(/~- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 m M MgC12, 10

m M 2-mercaptoethanol] containing 10/iM (p-amidinophenyl)methanesul- fonyl fluoride and 100 mM KCI This suspension is sonicated on ice 10 times each for 30 sec with 30-sec intervals to break the cells Postnuclear supernatant (PNS) is obtained by centrifugation of the homogenate at 1000g for 5 min at 4 ° The PNS is then centrifuged at 160,000g (Beckman SW40 rotor, 30,000 rpm) for 30 min at 4 ° About 10% of Rab5 is recovered

in the pellet and 90% in the supernatant, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) stained with Coomassie blue (Fig 1B, lanes 1-3) and by Western blot analysis using anti-Rab5 monoclonal antibody (data not shown) (see [27] in this volume) The main band of Rab5 in the pellet migrates slightly faster than that in the supernatant on S D S - P A G E This is an indication that Rab5 in the pellet is posttranslationaUy modified while the protein in the supernatant

is not a6 A further criterion to distinguish between the two forms is the interaction with R a b - G D I (see below) The reason why most of the Rab5

is recovered in cytosol may be due to limitations of the Rab GGTase and/

of fractions 4-24 (lanes 4-14) For details, see text

idopropyl)dimethylammonio]-l-propanesulfonic acid (CHAPS) (Sigma) with sonication for 10 sec on ice and is incubated for 1 hr at 4 ° on a rotating wheel The suspension is centrifuged at 160,000g for 30 min at 4 °, and the supernatant (4 ml, 10 mg protein) is loaded onto a Mono Q HR5/5 column (Pharmacia) equilibrated with degassed buffer A containing 0.6% (w/v) CHAPS (Fig 1A) After washing the column with 12 ml of the same buffer, proteins are eluted with buffer A containing 0.6% CHAPS and 1 M NaC1 Fractions (0.5 ml) are collected and analyzed by S D S - P A G E stained with Coomassie blue (Fig 1B) and immunoblotting using anti-Rab5 monoclonal antibody (data not shown) (see [27] in this volume) Most of Rab5 is detected in two peaks The first consists of the flow-through fractions (frac-

14 EXPRESSION, PURIFICATION, AND MODIFICATION [2] tions 4-11; about 20% recovery) and the second consists of the washing fractions (fractions 12-24, about 40% recovery), where Rab5 migrates slightly faster compared to the protein contained in the first peak on SDS- PAGE These fractions are further characterized for the presence of post- translationally modified Rab5 Since R a b - G D I has been shown to be active only on posttranslationally modified but not on unmodified Rab proteins, 5

we tested each fraction for R a b - G D I to inhibit GDP/GTP exchange as deduced by the binding of radiolabeled GTPTS 4 (Fig 1A) An aliquot (2/zl) of each fraction is incubated in the presence or in the absence of 5 /zM Rab-GDI, purified from overexpressing E coli as a His6-tagged protein (see [27] in this volume), in a buffer (20/zl) containing 20 m M HEPES/ KOH (pH 7.2), 10 m M EDTA, 5 m M MgC12, 1 mM dithiothreitol, and 1/zM [35S]GTPTS (20,000 cpm/pmol, DuPont-NEN) for 10 min at 30 ° Protein-bound [35S]GTPTS is measured by passing the reaction mixture through a nitrocellulose filter (0.45-/zm pore size, 2.5 cm diameter, BA85, Schleicher & Schuell) immediately after adding 3 ml of filtration buffer [20

m M tris[hydroxymethyl]aminomethane hydrochloride (pH 7.5), 100 mM NaCI, and 25 m M MgC12] After three washes with 3 ml filtration buffer, the filter is dried and the radioactivity is measured in 5 ml Ready Safe scintillation liquid (Beckman) using a Beckman LS 6000SC type scintillation counter Proteins in these fractions effectively bind [35S]GTPyS Although

R a b - G D I does not effect [35S]GTPyS binding to the proteins of fractions 4-11, it effectively inhibits [35S]GTPyS binding to the proteins of fractions 12-24, thus indicating that the second peak (fractions 12-24) Contains posttranslationally modified Rab5 The Rab5 protein recovered in fractions 4-11 may come from the contaminating cytosol and/or aggregated cytosol Rab5 The samples are analyzed by S D S - P A G E (12% acrylamide gel) stained with Coomassie blue (Fig 1B, lanes 4-14) Typically, about 200/zg

of highly purified posttranslationally modified Rab5 is obtained in frac- tions 12-24

Purification of Posttranslationally Unmodified Rab5 from Cytosol

of Sf9 Cells

The posttranslationally unmodified Rab5 is purified from the cytosol

of Rab5-expressing Sf9 cells by a one-step procedure using hydroxyapatite column chromatography Hydroxyapatite (Seikagakukogyo, Tokyo, Japan)

is swollen in distilled water and the fine particles are removed by changing the water every 30 min until the supernatant is clear Then, 1 ml of hydroxy- apatite is transferred onto a Poly-Prep chromatography column (Bio-Rad), followed by equilibration with buffer B (20 m M HEPES/KOH, pH 7.2, 5

m M MgCIz, 10 m M 2-mercaptoethanol) The cytosol (1 ml, 5 mg of protein)

[3] Rab9 PURIFICATION AND ISOPRENYLATION 15

is loaded onto the column After washing the column with 5 ml of buffer

B, the column is eluted with buffer B containing 0.6% CHAPS Fractions (0.5 ml) are collected, and 150/zg of unmodified Rab5 is eluted in fractions 2-8 Because of the high level of expression and the particular property

of Rab5 to be eluted by CHAPS, the purity is over 90% Purified unmodified Rab5 efficiently binds GTP and GDP but, as expected, R a b - G D I does not inhibit [35S]GTP3~S binding in the same assay mentioned earlier In this simple procedure, 3 mg of highly purified unmodified Rab5 can be expected from one preparation of the cytosol (20 ml)

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 rab proteins In addition, characterization

of purified mutant forms of Rab proteins can provide valuable information

to complement functional studies of Rab proteins in in vitro systems or in living cells

The pET expression system developed by Studier et al 1 is invaluable for the production of milligram quantities of specific proteins in E coli

Rab9 cDNA was subcloned into the pET8c plasmid, which places the cDNA under the control of a T7 RNA polymerase promoter The resulting expression vector, pET8c-Rab9, is transformed into the E coli strain BL21 (DE3), which expresses the T7 RNA polymerase gene under the control

of the lacZ promoter The addition of isopropyl-/3-D-thiogalactoside (IPTG) induces the synthesis of T7 RNA polymerase, which, when present at high levels, produces large amounts of Rab9 mRNA and thus large amounts of Rab9 protein

1 F Studier, A Rosenberg, J Dunn, and J Dubendorf, this series, Vol 185, p 60

Copyright © 1995 by Academic Press, Inc

[3] Rab9 PURIFICATION AND ISOPRENYLATION 15

is loaded onto the column After washing the column with 5 ml of buffer

B, the column is eluted with buffer B containing 0.6% CHAPS Fractions (0.5 ml) are collected, and 150/zg of unmodified Rab5 is eluted in fractions 2-8 Because of the high level of expression and the particular property

of Rab5 to be eluted by CHAPS, the purity is over 90% Purified unmodified Rab5 efficiently binds GTP and GDP but, as expected, R a b - G D I does not inhibit [35S]GTP3~S binding in the same assay mentioned earlier In this simple procedure, 3 mg of highly purified unmodified Rab5 can be expected from one preparation of the cytosol (20 ml)

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 rab proteins In addition, characterization

of purified mutant forms of Rab proteins can provide valuable information

to complement functional studies of Rab proteins in in vitro systems or in living cells

The pET expression system developed by Studier et al 1 is invaluable for the production of milligram quantities of specific proteins in E coli

Rab9 cDNA was subcloned into the pET8c plasmid, which places the cDNA under the control of a T7 RNA polymerase promoter The resulting expression vector, pET8c-Rab9, is transformed into the E coli strain BL21 (DE3), which expresses the T7 RNA polymerase gene under the control

of the lacZ promoter The addition of isopropyl-/3-D-thiogalactoside (IPTG) induces the synthesis of T7 RNA polymerase, which, when present at high levels, produces large amounts of Rab9 mRNA and thus large amounts of Rab9 protein

1 F Studier, A Rosenberg, J Dunn, and J Dubendorf, this series, Vol 185, p 60

Copyright © 1995 by Academic Press, Inc

16 EXPRESSION, PURIFICATION, AND MODIFICATION [31 Materials

IPTG, ampicillin, geranylgeranyl pyrophosphate, and [3H]geranylgera- nyl pyrophosphate (GGPP and [3H]GGPP, American Radiolabeled Chem- icals)

Pressure filtration cell (Amicon)

Q-Sepharose Fast Flow column (Pharmacia)

Sephacryl S-100 column (Pharmacia)

Buffers

Lysis buffer: 64 m M Tris-HCl (pH 8.0), 8 m M MgCI2,2 m M EDTA, 0.5

m M dithiothreitol (DTT), 10/zM GDP, 1 m M phenylmethylsulfonyl fluoride (PMSF), 10 m M benzamidine, 10/zg/ml leupeptin, 1 tzM pepstatin, 3/zg/ml aprotinin, and 1 m M NaNz

S-100 buffer: 64 m M Tris-HC1 (pH 8.0), 100 m M NaC1, 8 m M MgC12,

2 m M EDTA, 0.2 m M DTT, 10/.~M GDP, 1 m M PMSF, 10 m M benzamidine, and 1 m M NaN3

Procedures

Expression and Purification o f Rab9 Protein

The procedure was optimized for Rab9 purification based on a pre-

viously described method of Tucker et al 2

1 The cDNA of rab9 was cloned into the E coli expression vector, pET8c 1 The pET8c plasmid was linearized with BamHI, filled in using the Klenow fragment of DNA polymerase I, and cut with NcoI Both restriction

enzyme sites are located in the polylinker of pET8c A pGEM1-Rab9

2 j Tucker, G Sczakiel, J Feuerstein, J John, R Goody, and A Wittinghofer, EMBO J 5,

1351 (1986)

[3] Rab9 PURIFICATION AND ISOPRENYLATION 17

plasmid 3 was linearized with PstI and filled in with T4 DNA polymerase

A second NcoI digestion liberated a fragment containing the rab9 gene

The pET8c and Rab9 fragments were purified by agarose gel electrophoresis prior to ligation by standard procedures 4 The construct was confirmed by

restriction analysis and transformed into the E coli strain BL21

2 An overnight culture of BL21 + pET8c-Rab9wt is grown in LB + ampicillin (100/zg/ml) Five hundred milliliters of LB + ampicillin (100 /zg/ml) is inoculated with 5 ml of overnight culture and grown to a n OD60o

of 0.4-0.6 Induction is started by the addition of IPTG to a final concentra- tion of 0.4 mM Induction at 37 ° is performed for 3.5 hr before the cells are centrifuged for 5 min at 6000 rpm The supernatant is discarded, and the cell pellet is frozen in liquid N2 and stored at - 2 0 °

3 The bacterial pellet is resuspended in 15 ml ice-cold lysis buffer The cells are lysed by two passages through a French press at medium power with 1400 units pressure Subsequent steps are performed at 4 °

4 Protamine sulfate is added to a final concentration of 1 mg/ml and the suspension is stirred for 2 min The mixture is then centrifuged for 5 min at 16,000 rpm in a precooled Sorvall SS-34 rotor

5 The supernatant is loaded onto a 15-ml Q-Sepharose Fast Flow col- umn preequilibrated in lysis buffer and washed with 20 ml of lysis buffer Proteins are eluted with a 2 x 50-ml gradient of 0-200 mM NaC1 in lysis buffer and 2-ml fractions are collected

6 Alternate fractions (20/xl) are analyzed by polyacrylamide gel elec- trophoresis (12%) and proteins are visualized by Coomassie blue staining Rab9 protein is determined by size comparison with control Rab9 protein

on the stained gel The identity of the band is later confirmed by Western blot and GTP overlay

7 Fractions containing Rab9 are pooled, concentrated to a final volume

of 2 ml by pressure filtration in a stirred cell, and applied to a 240-ml Sephacryl S-100 column The column is run in S-100 buffer at a rate of about 20 ml per hour; 80 fractions of 2.5 ml are collected

8 Fractions containing Rab9 are pooled and concentrated by pressure filtration to a final concentration of 0.4-1.0 mg/ml Rab9 protein is either rapidly frozen in liquid nitrogen and stored at - 8 0 ° or stored in 40% glycerol

at - 2 0 °

Notes: (1) Rab9 protein has the unique property of being very efficiently

proteolysed at the carboxy terminus No commercially available protease

3 D Lombardi, T Soldati, M A Riederer, Y Goda, M Zerial, and S R Pfeffer, E M B O J

12, 677 (1993)

4 j Sambrook, E Fritsch, and T Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd

ed Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1989

18 EXPRESSION, PURIFICATION, AND MODIFICATION [3]

inhibitor was found to inhibit this proteolytic step The only significant way

to increase the yield of full-length Rab9 protein is to work at 4 ° and to work as fast as possible Rab9 and the unknown protease are resolved on the Q-Sepharose column, where the protease activity elutes at a higher salt concentration relative to Rab9 protein (2) The Rab9S21N mutant is much less soluble when expressed in E coli The modifications described below increase the pool of soluble Rab9S21N and permit the purification of small quantities of Rab9S21N protein 5

Results

1 Induction: After induction for 3.5 hr, the 26-kDa Rab9 polypeptide

is clearly detectable in cell extracts subjected to S D S - P A G E and Coomassie blue staining The identity of the 26-kDA protein is confirmed by immu- noblot analysis using a Rab9-specific antibody In addition, the expressed protein binds GTP as determined by the [o~-32p]GTP overlay of proteins resolved by S D S - P A G E and transferred to nitrocellulose

2 Ion exchange: Soluble fractions are subjected to ion-exchange chro- matography on a column of Q-Sepharose The [a-32p]GTP overlay of the collected Q-Sepharose fractions reveals two peaks of GTP-binding activity Immunoblot analyses show that both peaks contain Rab9-immunoreactive material: immunoreactive material in the first peak migrates as a 26-kDa polypeptide and, in the second peak, as a 22-kDa species The larger poly- peptide comigrates with Rab9 protein present in 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 and thus does not contaminate the final preparation In summary, Rab9 elutes from the Q-Sepharose at 120 m M NaC1; the Q-Sepharose chromatography results in a sevenfold purification relative to the initial cell lysate (Table I)

3 Gel filtration: The pooled Q-Sepharose fractions are concentrated

by pressure filtration and are subjected to gel filtration using Sephacryl S-100 The Rab9 protein migrates with a retention coefficient of 0.6 The S-100 column results in an additional -fourfold purification (Table I)

4 In summary, the two-step purification yields a 27-fold enrichment of Rab9 protein and a final preparation that is ->95% pure One gram of cell paste yields 0.8 mg of Rab9 Rab9 preparations are typically -90% active

5 M A Riederer, T Soldati, A D Shapiro, J Lin, and S R Pfeffer, J Cell Biol 125, 573 (1994)

[3] Rab9 PURIFICATION AND ISOPRENYLATION 19

TABLE I PURIFICATION OF Rab9 PROTEIN

Total nucleotide Total Total binding Specific

protein volume activity activity Yield Purification Fraction (mg) (ml) (nmol) (nmol/mg) (%) (-fold)

as judged by the extent of [ot-32p]GTP binding relative to applied protein 6

No loss of binding activity has been detected after >2 months of storage

at - 2 0 ° in S-100 column buffer containing 40% glycerol

Notes: (1) We have also created a Rab9 protein that possesses a different carboxy-terminal tetrapeptide which serves as a signal for isoprenylation

by prenyltransferase I The resulting R a b 9 - C L L L is not degraded during the purification process, suggesting that a carboxypeptidase initiates the proteolytic processing and preferentially cleaves Rab9 protein terminating

in CC (2) Other Rab9 mutant proteins: After induction for 6 hr at 30 °, a small pool of Rab9S21N is soluble and can be purified using the same procedure employed for Rab9 In contrast, Rab9140M and Rab9N127I are insoluble under conditions that allow purification of Rab9S21N Attempts

to solubilize these mutant proteins in 6 M guanidinium-chloride followed

by dilution have not been successful (3) Rab7 can be expressed in E coli

and purified using the identical procedure described earlier Rab7 appears

to be resistant to proteases and is eluted from the Q-Sepharose column at

70 mM NaC1

Isoprenylation of Rab9 in Vitro

Small-Scale in Vitro Prenylation

Small-scale prenylation reactions are extremely useful either to test in vitro the prenylatability of a Rab protein or mutant thereof or, alternatively,

to optimize the conditions prior to preparative scale incubations A standard 50-/zl reaction contains 5 ng Rab9, 50 ng GGPP, and 1.5 mg/ml crude ClIO cytosol The buffer conditions are similar to those used for in vitro endosome

6 A D Shapiro, M A Riederer, and S R Pfeffer, J Biol Chem 268, 6925 (1993)

20 EXPRESSION, PURIFICATION, AND MODIFICATION [3]

to TGN transport 7 22 mM HEPES-KOH, pH 7.2, 20 mM Tris-HC1, 116

mM KC1, 4.3 mM magnesium acetate + MgC12), 2 m M DTT, and 0.2 mM GDP, plus a protease inhibitor cocktail and an ATP-regenerating system After incubation at 37 ° for 1 to 2 hr, the prenylation reactions are clarified

by ultracentrifugation at 300,000g for 10 min in a TLA100.2 rotor (Beck- man) and analyzed by 12.5% S D S - P A G E and anti-Rab9 immunoblotting

As prenylated Rab9 migrates slightly faster than the unprenylated starting material, the efficiency of the prenylation reaction can be easily monitored Alternatively, if no molecular size shift is expected, or for precise quantita- tion analysis, small-scale reactions should include 1/xM GGPP and 0.1/xM [3H]GGPP, and be followed by S D S - P A G E and fluorography

Another small-scale prenylation assay used to assess prenylatability of

a construct is based on the cell-free translation of an in vitro-transcribed Rab

eDNA Commercially available rabbit reticulocyte lysate (e.g., Promega) is gel filtered and therefore does not contain enough endogenous GGPP to ensure prenylation of newly translated proteins Efficient prenylation can

be achieved by adding 10 tzM GGPP in the in vitro translation reaction

containing [35S]methionine (as judged then by a molecular size shift after

S D S - P A G E and autoradiography analysis) or 1/zM GGPP and 0.1/zM [3H]GGPP in reactions carried out with unlabeled amino acids (as judged

by incorporation of radioactivity in the translation product analyzed by

S D S - P A G E and fluorography)

Preparative in Vitro Prenylation

In a standard 0.5-ml reaction, 1 tzg of purified Rab9 (100 nM) is preny- lated in the presence of 5.6 mg/ml of crude Chinese hamster ovary (CHO) cytosol (prepared as described in Goda and Pfeffer 7) and 10 tzM of geranyl- geranyl pyrophosphate (GGPP, American Radiolabeled Chemicals, Inc)

by incubation for 1 hr at 37 ° Preparative prenylation of Rab9 protein is usually about 50-80% efficient 8 Separation of the prenylated Rab9 from nonreacted or degraded material by Sephacryl S-100 gel filtration chroma- tography (see below) is facilitated by the fact that prenylated Rab proteins associate with GDP dissociation inhibitor (GDI) and hence fraetionate at

~80 kDa, whereas the other products will elute around 20-30 kDa

Gel Filtration Chromatography and Fraction Analysis Samples are ana-

lyzed on a 50-ml Sephacryl S-100 (Pharmacia) column equilibrated and eluted in S-100 buffer (64 m M Tris/HC1, pH 8, 100 m M NaC1, 8 m M MgCI2,

2 mM EDTA, 0.2 m M DTI', 10 tzM GDP, and 1 m M PMSF) Forty 0.4-

ml fractions are collected; alternate fractions are subjected to 12.5% SDS-

7 y G o d a and S R Pfeffer, Cell (Cambridge, Mass.) $5, 309 (1988)

s T Soldati, M A Riederer, and S R Pfeffer, Mol Biol Cell 4, 425 (1993)

[4] CHARACTERIZATION OF TYPE-II GGTase 21

P A G E and conventional immunoblotting Rab9 protein is detected using rabbit or mouse antibodies raised against native, recombinant Rab9 pro- tein 8 Detection of GDI is carried out using affinity-purified antibodies raised against purified Rab3A-GDI 8 Secondary antibodies are either goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad) All antibodies are used at 1:1000 dilution; antigen-antibody complexes are detected by enhanced chemiluminescence (ECL, Amer- sham) Quantitation of ECL signals on X-ray films (Kodak) is carried out using a densitometric scanner (Model 300 A, Molecular Dynamics) or a Phosphorlmager system (Molecular Dynamics)

in the cytosol, but become membrane bound after undergoing posttransla- tional modification Mutations that block the membrane attachment of these proteins result in a block in secretion, a,5 Thus, the membrane association of Yptlp and Sec4p is crucial for their function

The ability of small GTP-binding proteins to bind to membranes is conferred by the addition of geranylgeranyl, a 20-carbon isoprenoid deriva-

1 M Zerial and H Stenmark, Curr Opin Cell Biol 5, 613 (1993)

2 A Salminen and P J Novick, Cell (Cambridge, Mass.) 49, 527 (1987)

3 N Segev, J Mulholland, and D Botstein, Cell (Cambridge, Mass.) 52, 915 (1988)

4 G Rossi, Y Jiang, A P Newman, and S Ferro-Novick, Nature (London) 351, 158 (1991)

s R Li, C Havel, J A Watson, and A W Murray, Nature (London) 366, 82 (1993)

Copyright © 1995 by Academic Press, Inc

[4] CHARACTERIZATION OF TYPE-II GGTase 21

P A G E and conventional immunoblotting Rab9 protein is detected using rabbit or mouse antibodies raised against native, recombinant Rab9 pro- tein 8 Detection of GDI is carried out using affinity-purified antibodies raised against purified Rab3A-GDI 8 Secondary antibodies are either goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad) All antibodies are used at 1:1000 dilution; antigen-antibody complexes are detected by enhanced chemiluminescence (ECL, Amer- sham) Quantitation of ECL signals on X-ray films (Kodak) is carried out using a densitometric scanner (Model 300 A, Molecular Dynamics) or a Phosphorlmager system (Molecular Dynamics)

in the cytosol, but become membrane bound after undergoing posttransla- tional modification Mutations that block the membrane attachment of these proteins result in a block in secretion, a,5 Thus, the membrane association of Yptlp and Sec4p is crucial for their function

The ability of small GTP-binding proteins to bind to membranes is conferred by the addition of geranylgeranyl, a 20-carbon isoprenoid deriva-

1 M Zerial and H Stenmark, Curr Opin Cell Biol 5, 613 (1993)

2 A Salminen and P J Novick, Cell (Cambridge, Mass.) 49, 527 (1987)

3 N Segev, J Mulholland, and D Botstein, Cell (Cambridge, Mass.) 52, 915 (1988)

4 G Rossi, Y Jiang, A P Newman, and S Ferro-Novick, Nature (London) 351, 158 (1991)

s R Li, C Havel, J A Watson, and A W Murray, Nature (London) 366, 82 (1993)

Copyright © 1995 by Academic Press, Inc

22 EXPRESSION, PURIFICATION, AND MODIFICATION [4] tive that is attached to carboxy-terminal cysteine moieties (CC o r CXC) 6,7

T h e prenyltransferase that p e r f o r m s this task has b e e n identified in yeast and m a m m a l i a n cells 6-1° T w o other prenyltransferases, the farnesyltrans- ferase and the type I geranylgeranyltransferase ( G G T a s e I), have b e e n shown to be heterodimers 11-14 T h e s e enzymes modify proteins that termi- nate in a C A A X box (C, cysteine; A, aliphatic amino acid; X, any amino acid) T h e e n z y m e that modifies Y p t l p and Sec4p, called R a b or t y p e - I I geranylgeranyltransferase ( G G T a s e II), differs f r o m the other prenyltrans- ferases in its subunit structure G G T a s e II is a multisubunit e n z y m e that consists of two separate components: a catalytic c o m p o n e n t (originally

n a m e d c o m p o n e n t B) and an accessory subunit (also called c o m p o n e n t A) 6-1° T h e catalytic c o m p o n e n t is a h e t e r o d i m e r c o m p o s e d of a a n d / 3 subunits 6,7 T h e 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 c o m p o - nent of the enzyme 8,l° In the yeast S a c c h a r o m y c e s cerevisiae, the/3 subunit

of the catalytic c o m p o n e n t is a 36.6-kDa hydrophilic protein that is e n c o d e d

by the B E 7 2 gene 4,7 T h e 35-kDa a subunit is the p r o d u c t of the B E T 4

gene (originally called M A D 2 ) 2'5'7 These two subunits f o r m a stable hetero-

d i m e r that is active 7 T h e third subunit, which serves as the escort protein,

is a 66-kDa hydrophilic protein that is encoded by the M R S 6 gene 8 In vivo

and in vitro studies have d e m o n s t r a t e d that all three subunits are required for m a x i m a l prenylation activity 8 This chapter presents the e x p e r i m e n t a l details for studying the yeast G G T a s e II In addition, the c h a p t e r describes

a system for expressing its subunits in bacterial cells and the techniques

e m p l o y e d for reconstituting G G T a s e I I activity in vitro

D e t e r m i n a t i o n of G G T a s e II Activity in Y e a s t L y s a t e s

O u r studies on G G T a s e II activity in yeast are initiated by establishing

an in vitro assay This assay enables us to dissect G G T a s e I I activity further

in terms of the substrate specificity of the e n z y m e and the subunit composi-

6 M C Seabra, J L Goldstein, T C Sudhof, and M S Brown, J Biol Chem 267,14497 (1992)

7 y 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 Goldstein, Cell (Cambridge, Mass.) 70, 1049 (1992)

10 D 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)

11 M L Mayer, B E Caplin, and M S Marshall, J Biol Chem 267, 20589 (1992)

12 B He, P Chen, S.-Y Chen, K L Vancura, S Michaelis, and S Powers, Proc Natl Acad Sci U.S.A 88, 11373 (1991)

13 j F Moomaw and P J Casey, J Biol Chem 267, 17438 (1992)

14 y Reiss, M C Seabra, M S Brown, and J L Goldstein, Biochem Soc Trans 20, 487 (1992)

[4] CHARACTERIZATION OF TYPE-n GGTase 23 tion T h e assay is p e r f o r m e d by incubating unprenylated substrate with a radiolabeled prenyl p y r o p h o s p h a t e precursor in the presence of a soluble yeast cell extract Radiolabeled protein is detected by a filter-binding assay

or by electrophoresing the reaction product on a S D S - p o l y a c r y l a m i d e gel Once the assay conditions for G G T a s e - I I are defined, mutant extracts are

p r e p a r e d and analyzed for a defect in activity The assay is p e r f o r m e d with recombinant Y p t l and Sec4 proteins, which are purified as described before, 15,16 radiolabeled geranylgeranyl pyrophosphate, and yeast extracts (see below) T h e extracts are p r e p a r e d by a modification of previously published protocols 17,18

P r e p a r a t i o n o f S o l u b l e Y e a s t Extracts

Concentrated yeast lysates are p r e p a r e d by glass bead lysis of cells

B e f o r e use, the beads (0.5 mm, Biospec Products, Inc.) are washed succes- sively with the following solutions: 1% TritonX-100 ( 2 x ) , 95% ethanol ( 2 x ) , 6 N HC1 ( 2 x ) , 6 N HNO3 ( 2 x ) , and double-distilled water until the water is p H 6.0 B e t w e e n each wash the beads are rinsed 2 x with double- distilled water T h e washed beads are dried in a oven and stored at 4 ° Yeast cells are grown overnight at 25 ° to late log phase in YP medium (10 g/liter yeast extract, 20 g/liter p e p t o n e ) that is supplemented with 2% glucose T h e cells (150 OD60o units) are pelleted in 50-ml conical tubes, washed with 5 ml of sterile water, resuspended in 1 ml of ice-cold lysis buffer [100 m M MES (4-morpholineethanesulphonic a c i d ) - N a O H , p H 6.5, 0.1 m M MgCI2,0.1 m M E G T A , 1 m M 2-mercaptoethanol, and 2 m M phenyl- methyl-sulfonyl fluoride (PMSF)], and lysed in the presence of 6 g of glass beads Cells are lysed by 10 repetitive cycles of vortexing (10 sec) and cooling on ice (30 sec) T o remove the beads and u n b r o k e n cells, the lysate

is centrifuged at 500g (1450 rpm) for 5 min at 4 ° in a B e c k m a n tabletop centrifuge T h e supernatant is recentrifuged at 120,000g for 1 hr at 4 ° in a SW 50.1 r o t o r (5 x 41-mm ultracentrifuge tube) T h e high-speed supernatant is separated from the pellet, and the protein concentration of the soluble fraction (30-40 mg/ml) is estimated using the Bradford assay 19 This soluble extract is aliquoted, frozen in liquid nitrogen, and stored at - 8 0 ° When

15 p Novick, M D Garrett, P Brennwald, and A K Kabcenell, this series, Vol 219, p 352

~6 p Wagner, C M T Molenaar, A J G Rauh, R BrOkel, H D Schmitt, and D Gallwitz,

EMBO J 6, 2373 (1987)

17 S L Moores, M D Schaber, S D Mosser, E Rands, M B O'Hara, V M Garsky, M S Marshall, D L Pompliano, and J B Gibbs, J Biol Chem 266, 14603 (1991)

18 L E Goodman, S R Judd, C C Farnsworth, S Powers, M H Gelb, J A Glomset, and

F Tamanoi, Proc Natl Acad Sci U.S.A 87, 9665 (1990)

19 M M Bradford, Anal Biochem 72, 248 (1976)

24 EXPRESSION, PURIFICATION, AND MODIFICATION [4] mutant extracts are examined, the cells are grown overnight at 24 ° and then shifted to 37 ° (restrictive temperature) for 1 hr prior to lysis

GGTase H Assay Using Yeast Lysates

The GGTase II assay is performed using purified recombinant Yptlp ( ~ 3 0 / z M ) as a protein substrate and [3H]geranylgeranyl pyrophosphate (GGPP) triammonium salt (-18,000 dpm/pmol; American Radiolabeled Chemicals, Inc.) as a lipid donor The purification of recombinant Yptlp from E coli cells has been reported before, 16 and the prenylation assay we use has been established by modifying a protocol that has been described previously 17'18 Briefly, a standard 50-/zl reaction contains the following reagents: 50 mM KPi, pH 7.4, 5 mM DTT, 10 mM MgCI2, 0.4/~M recombi- nant Yptlp protein, 0.8 ~M radiolabeled GGPP (15 Ci/mmol) and water

up to 50/~1 All reactions are conducted in an Eppendorf tube The sample

is first preincubated for 10 rain at 30 ° before 50/zg of a soluble extract is added to the mixture The sample is then incubated at 30 ° for an additional

30 min The product of the reaction is examined by SDS gel (12.5%) electrophoresis or a filter assay To perform the filter assay, the reaction

is terminated by transferring the sample to a tube containing 1 ml of 1 N HC1 (in ethanol) Subsequent to a 15-min incubation at room temperature,

2 ml of ethanol is added and the reaction mix is filtered through a 24-mm

G F / A glass microfiber filter The filter is washed with 20 ml of ethanol, dried, and counted in a scintillation counter Background is calculated as the amount of [3H]GGPP that binds to the filter in the absence of Yptlp GGTase II activity is expressed as picomoles of Yptlp geranylgeranylated per minute per milligram of soluble yeast lysate

Upon determining the candidate genes that encode the three subunits

of the yeast GGTase II, it was possible to introduce each of them into different bacterial strains and reconstitute GGTase II activity in vitro We chose Escherichia coli to express the recombinant subunits because it is devoid of prenylation activity and easy to manipulate In this way, the measured activity directly reflects the reconstitution of the enzyme complex

Preparation o f Expression Vectors

To express BET2 in E coli, it is cloned into a p U C l l 8 expression vector This vector utilizes an inducible lac promoter that controls the expression of/3-galactosidase (encoded by the lacZ gene) Plasmid p U C l l 8 contains

a polylinker region at the 5' end of the lacZ gene that is used to clone a

[4] CHARACTERIZATION OF TYPE-II GGTase 25 target gene in the correct reading frame The resulting hybrid protein, which is induced with isopropylthio-/3-o-galactoside (IPTG), contains a portion of/3-galactosidase (N terminus) fused to the target gene product The lac promoter induces a low level of expression of the fusion protein,

therefore minimizing the formation of insoluble aggregates

To clone BET2 into pUC118, we engineered a restriction site at the start

of the gene using the polymerase chain reaction (PCR) This construction maintains BET2 in the correct reading frame Two oligonucleotides are

used to amplify the gene The first one incorporates a KpnI site just behind

the initial A T G and the second introduces a SalI site downstream from

the termination codon The PCR product is then digested with KpnI and SalI and is ligated into the KpnI and SalI sites of p U C l l 8 to yield pSFN171

This plasmid encodes Bet2p (lacking the start methionine) fused to the first nine amino acids of/3-galactosidase The construct is then transformed into JM101 cells and the transformants are selected at 37 ° on LB plates (10 g tryptone, 5 g yeast extract, 5 g NaCI, and 15 g agar per liter) containing 100/.~g/ml of ampicillin A single transformant, containing the correct con- struction, is grown overnight at 37 ° in 5 ml of LB medium that is supple- mented with 100/xg/ml ampicillin Plasmid D N A is purified from the cell culture and stored at - 2 0 ° for subsequent cotransformation experiments

A frozen stock is also prepared from the overnight cell culture and is stored

at - 8 0 ° for future use

Plasmid pBC-KS (Stratagene) is chosen to express the BET4 gene This

plasmid employs the same lacZ expression system as pUC118 However, un-

like p U C l l 8 , it contains the gene conferring chloramphenicol drug resis- tance The BET4 gene is amplified by PCR using two oligonucleotides A KpnI site is introduced at the 5' end of one of the oligonucleotides This site

overlaps with the initiation codon of BET4 placing it in-frame with 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 its start methionine) Placing this portion of/3- galactosidase at the N terminus of Bet4p, however, interferes with its func- tion To reduce this interference, the sequence upstream from the KpnI site

on pSFN172 is replaced with a sequence from p U C l l 8 This is done by excis- • ing the region between AfllII and KpnI from pSFN172 and replacing it with

a 0.34-kb AfllII-KpnI fragment from p U C l l 8 Because the coding sequence

upstream of the KpnI site in pUC118 is shorter than that of pBC-KS, 11 amino acids are deleted from the/3-galactosidase portion of the fusion product (/3- galactosidase-Bet4p-2) The resulting plasmid (pSFN173) is amplified by

26 EXPRESSION, PURIFICATION, AND MODIFICATION [4] transforming it into JM101 cells Since pBC-KS contains the chloramphenicol resistance gene, LB plates containing chloramphenicol (25/zg/ml) are used

to select the transformants Overnight cell cultures are prepared from the transformants and plasmid DNA is extracted

Coexpression of Bet2p and Bet4p in E coli and Preparation of

Cell Lysates

In order to coexpress Bet2p and Bet4p in E coli, plasmids pSFN171 and pSFN173 are transformed simultaneously into JM101 These vectors contain two different drug resistance genes that allow us to select for both vectors at the same time Freshly made competent cells, prepared as described before, 2° are recommended for optimal transformation and expression To cotrans- form JM101 cells, 200 ng of pSFN171 and 200 ng of pSFN173 are incubated with 50/zl of competent cells on ice for 30 min The reaction mixture is then shifted to 42 ° for 2 min and reincubated on ice for another 2 min before adding 200/zl of LB medium The mixture is shifted to 37 ° and after a 45-min incuba- tion with gentle shaking, the sample is spread onto a LB plate that contains ampicillin (100/xg/ml) and chloramphenicol (25/zg/ml) After the plates are incubated for 12-18 hr at 37 °, 10-20 fresh transformants are inoculated into

a 250-ml conical flask containing 25 ml of LB medium that is supplemented with 100/~g/ml ampicillin and 25/xg/ml of chloramphenicol The cells are grown at 37 ° for 1-2 hr with vigorous agitation until the cell density at OD60o

is between 0.4 and 0.6 The expression of Bet2p and Bet4p is then induced with IPTG (0.4 mM) during a 4-hr incubation Subsequently, the culture is chilled on ice for 10 min and the cells are pelleted in a Beckman centrifuge during a spin at 4000 rpm (4°) The pellet is resuspended in a ml of ice-cold lysis buffer (100 mM MES-NaOH, pH 6.5, 0.1 mM MgC12, 0.1 mM EGTA,

1 mM 2-mercaptoethanol) and transferred into a 1.5-ml Eppendorf tube The cells are washed once with ice-cold lysis buffer before they are stored at - 8 0 °

ml Eppendorf tube, and the beads are rinsed with 0.3 ml of !ysis buffer The wash is combined with the supernatant The cell lysate is then centrifuged at 2o j Sarnbrook, E F Fritsch, and T Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd ed Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1989

[41 CHARACTERIZATION OF TYPE-II GGTase 27 top speed in a Eppendorf microfuge for 15 min at 4 ° to remove insoluble aggregates The supernatant is collected, and the protein concentration (usually about 20-25 mg/ml) is determined using the Bradford assay 19 The supernatant is aliquoted (50 tzl) into 0.5-ml Eppendorf tubes, frozen in liquid nitrogen, and stored at - 8 0 ° Because of the low level of expression, Bet2p and Bet4p are not detected on Coomassie blue-stained SDS- polyacrylamide gels However, the activity of the complex is measured directly using the filter-binding assay described earlier The presence of expressed Bet2p and Bet4p is also confirmed by immunoblots using anti- Bet2p and anti-Bet4p sera

Expression of Recombinant Mrs6p in E coli and Preparation of

Cell L ysates

Mrs6p is also expressed in E coli by utilizing the lacZ fusion expression

system of p U C l l 8 To clone the gene into pUC118, PCR is employed to generate a KpnI site at the start of MRS6 This placess MRS6 in-frame

with lacZ A PvuII site is also introduced at the 3' end of MRS6 about 100

bp downstream from the stop codon The PCR product is digested with

KpnI and PvuII and inserted into the KpnI and HincII sites of pUC118

The resulting construct (pSFN260) is used to transform JM101 cells Trans- formants containing the correct construction are grown overnight at 37 ° in

5 ml of LB medium that is supplemented with 100 ~g/ml of ampicillin The cell cultures are diluted 1 : 100 into 25 ml of the same medium and grown for 2-3 hr at 37 ° in a 250-ml flask When the OD600 is between 0.4 and 0.6, expression is initiated by the addition of IPTG (final concentration, 0.4 mM) After a 4-hr incubation at 37 °, the cells are harvested and lysed with glass beads using the protocol described earlier The lysate is subjected to centrifugation at 12,000g for 15 min at 4 ° and the supernatant is aliquoted (50 ~1) into 0.5-ml Eppendorf tubes, frozen in liquid nitrogen, and stored

at - 8 0 ° Since Mrs6p is not readily visualized on Coomassie blue-stained SDS-polyacrylamide gels, prenylation assays are performed As Mrs6p itself does not have GGTase II activity, its activity is measured through its ability to stimulate the prenylation activity of the Bet2p/Bet4p complex This assay is described below

In Vitro Reconstitution of GGTase H Activity Using

Recombinant Subunits

In performing in vitro prenylation assays, we use [3H]GGPP as a lipid

donor and recombinant Yptlp as a protein substrate However, similar results are also obtained when Sec4p serves as a substrate Activity is determined by measuring the amount of [3H]GGPP that is trapped onto a

28 EXPRESSION, PURIFICATION, AND MODIFICATION [4] Whatman GF/A glass microfiber filter The assay used to reconstitute yeast GGTase II activity differs in some respects from the standard prenylation assay Typically, a 50-/zl reaction contains 25/zg of lysate (20-25 mg/ml) prepared from cells expressing Mrs6p, 0.4/zM recombinant Yptlp, 50 mM Tris-HCl, pH 7.5, 10 mM MgC12, 5 mM dithiothreitol, and 0.8 /xM [3H]GGPP (18,000 dpm/pmol) After a 10-min incubation at 30 °, the preny- lation assay is initiated by the addition of 50/zg (concentration, 20-25 mg/ ml) of lysate prepared from cells that coexpress Bet2p and Bet4p The mixture is incubated for 30 min at 30 ° before the reaction is terminated When the filter assay is employed, the reaction is performed in a 6-ml glass tube and is terminated by the addition of 1 ml 1 N HC1 (diluted from 12

N with ethanol) If autoradiography is used to visualize the prenylated product, the reaction mix is incubated in a 1.5-ml Eppendorf tube and 50 /xl of 2x SDS sample buffer is used to terminate the assay (see Fig 1) Geranylgeranyltransferase activity is detected in extracts expressing Bet2p/Bet4p by assaying 20-50 /xg of lysate In a standard prenylation assay, 50/zg of lysate is assayed for geranylgeranyltransferase activity The

in bacteria Ram2p is the c~ subunit of the farnesyltransferase and GGTase I prenyltrans- ferases 12,18

[4] CHARACTERIZATION OF TYPE-II GGTase 29 reaction conditions have been described earlier In the absence of Mrs6p, coexpressed Bet2p/Bet4p displays a low level of GGTase II activity that

is detected by employing the filter assay The activity measured is largely

a consequence of the binding of [3H]GGPP onto the Bet2p/Bet4p complex

We have found that the coexpression of Bet2p and Bet4p is critical for the reconstitution of activity since no activity is detected when each of the subunits are expressed separately and then mixed together Two different /3-galactosidase-Bet4p fusion proteins have been constructed One contains the first 20 amino acids of/3-galactosidase fused to Bet4p (/3-galactosidase- Bet4p-1) This fusion protein forms a complex with Bet2p; however, in the presence of Mrs6p, a dramatic increase in prenylation activity does not ensue The deletion of 11 amino acids from this hybrid protein results in

a significant increase in prenylation activity This finding suggests that the N-terminal portion of/3-galactosidase-Bet4p-1 may disrupt the interaction between Mrs6p and the Bet2p/Bet4p complex However, further deletion

of the fl-galactosidase portion of the fusion protein does not lead to any increase in prenylation activity

in which the carboxy-terminal cysteine residues (CC) of Yptlp were re- placed 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 Reconstitution of GGTase II activity in vitro not only confirms the identity

of its three subunits (see Fig 1), but also provides an enriched source of this enzyme The efficiency of the reconstituted enzyme should make it possible to geranylgeranylate large quantities of recombinant Rab proteins

in vitro This in turn will facilitate the biochemical and molecular analysis

of these proteins

Acknowledgments

This work was supported by grants awarded to S F.-N from the National Cancer Institute

S F.-N is an associate investigator of the Howard Hughes Medical Institute

30 EXPRESSION, PURIFICATION, AND MODIFICATION [5]

a s e s 1'4 Two of these enzymes transfer prenyl groups to cysteines that are part of a COOH-terminal C A A X box sequence, where C is cysteine; A, aliphatic amino acid; and X, any amino acid C A A X farnesyltransfer-

a s e 5-7 typically recognizes C A A X boxes with methionine or serine at the

X position Substrate proteins include Ras as well as non-Ras proteins such

as nuclear lamins and at least two retinal proteins, the y subunit of trans- ducin and rhodopsin kinase C A A X GG transferase (or GG transferase 1) 8-1° recognizes C A A X boxes ending in leucine Its substrates include many members of the Ras superfamily, including Rho, Rac, Rap, and Ral as well as the 3~ subunits of heterotrimeric G proteins The third enzyme is designated Rab GG transferase (Rab GGTase or GG transferase 11) 1l 14

1 M S Boguski and F McCormick, Nature (London) 366, 643 (1993)

2 j A Glomset, M H Gelb, and C C Farnsworth, Curt Opin Lipidol 2, 118 (1991)

3 j L Goldstein and M S Brown, Nature (London) 343, 425 (1990)

4 M S Brown and J L Goldstein, Nature (London) 366, 14 (1993)

5 y Reiss, J L Goldstein, M C Seabra, P J Casey, and M S Brown, Cell (Cambridge,

Mass.) 62, 81 (1990)

6 y Reiss, S J Stradley, L M Gierasch, M S Brown, and J L Goldstein, Proc Natl Acad Sci U.S.A 88, 732 (1991)

7 S L Moores, M D Schaber, S D Mosser, E Rands, M B O'Hara, V M Garsky, M S

Marshall, D L Pompliano, and J B Gibbs, J Biol Chem 266, 14603 (1991)

8 M C Seabra, Y Reiss, P J Casey, M S Brown, and J L Goldstein, Cell (Cambridge,

Mass.) 65, 429 (1991)

9 j F M o o m a w and P J Casey, J Biol Chem 267, 17438 (1992)

10 K Yokoyama and M H Gelb, J Biol Chem 268, 4055 (1993)

11 M C Seabra, J L Goldstein, T C Stidhof, and M S Brown, J Biol Chem 267,14497 (1992)

12 M C Seabra, M S Brown, C A Slaughter, T C S0dhof, and J L Goldstein, Cell

(Cambridge, Mass.) 70, 1049 (1992)

13 D 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)

Copyright © 1995 by Academic Press, Inc

METHODS IN ENZYMOLOGY VOL 257 All rights of reproduction in any form reserved

[5] ENZYMES FOR PRENYLATION OF R a b PROTEINS 31 Its only identified substrates are Rab proteins In contrast to the other two

prenyltransferases, Rab GGTase does not recognize a C A A X box, but

rather it recognizes an as yet unidentified structural feature that is common

to all Rab proteins

Geranylgeranylation of Rab proteins is a complex enzymatic reaction that requires a Rab escort protein (REP) and Rab GGTase u-14 Rab GGTase is an od/3 heterodimer that transfers GG groups to cysteines at

or near the COOH termini of Rab proteins The target COOH-terminal sequences include CysCys, Cys X Cys, or CysCysXX, where X is any amino acid GG groups can be added to one or both of the cysteine residues 2 The 60-kDa ot and 38-kDa/3 subunits of Rab GGTase show amino acid

sequence homology to the corresponding subunits of the C A A X prenyl- transferases 4 However, unlike the C A A X prenyltransferases, which lack

REP components, Rab GGTase is inactive in the absence of REP 12-14 REP functions by binding newly synthesized unprenylated Rab proteins and presenting them to the Rab GGTase, which in turn transfers the GG moiety from geranylgeranyl pyrophosphate (GGPP) to Rab After transfer, the prenylated Rab remains complexed with REP and dissociates only when

it is removed by a postulated but yet unidentified Rab acceptor protein 13,15 Two distinct REP proteins, REP-1 and REP-2, have been identified, m s Rat REP-1 and human REP-2 are 70% identical and migrate as 95-kDa proteins on S D S - P A G E even though their predicted molecular masses are 72,545 and 74,064 Da, respectively The human gene for REP-1, located

on the X chromosome, is defective in patients with choroideremia, a form

of retinal degeneration 12'16'~7 The gene for REP-2 lacks introns and is found

on chromosome 1 TM Lymphoblasts derived from choroideremia patients have an 80% reduction in REP activity 19 The residual 20% of activity has

been attributed to REP-2 In vitro, REP-2 is equally as effective as

REP-1 in facilitating the covalent attachment of GG groups to several Rab proteins, including R a b l A (CysCys), Rab5A (CysCysSerAsn), and Rab6

14 S A Armstrong, M C Seabra, T C Siidhof, J L Goldstein, and M S Brown, J BioL Chem 268, 12221 (1993)

15 F P M Cremers, S A Armstrong, M C Seabra, M S Brown, and J L Goldstein, J

18 F P M Cremers, C M Molloy, D J R van de Pol, J A J M van den Hurk, I Bach,

A H M Geurts van Kessel, and H.-H Ropers, Hum Mol Genet 1, 71 (1992)

19 M C Seabra, M S Brown, and J L Goldstein, Science 259, 377 (1993)

32 EXPRESSION, PURIFICATION, AND MODIFICATION [5] (CysSerCys), but it is severalfold less active toward substrates of the neural- specific Rab3 (CysAlaCys) subfamily 15 Both proteins are expressed in all tissues examined so far, as determined by Northern blots

To date, only a single Rab GGTase heterodimer has been identified This catalytic subunit functions equally well with REP-1 or REP-2 The a and/3 subunits are unstable in the absence of each other Therefore, high level production of the catalytic moiety requires simultaneous coexpression

of the a and/3 subunit c D N A s ) 4

This chapter describes techniques for expression and purification of Rab GGTase and REP in insect Sf9 (Spodopterafrugiperda, fall armyworm ovary) cells using a baculovirus expression system that allows the production

of milligram quantities of active components for prenylation of Rab pro- teins The purified components can be used in vitro to prenylate all Rab proteins tested so far, regardless of the cysteine motif present at the

is flanked by viral recombination sequences The vector is cotransfected into Sf9 cells with viral DNA, and through a homologous recombination event the cDNA of interest is inserted into the viral genome When allowed

to infect Sf9 cells, the recombinant baculovirus will produce large quantities

of the desired protein The method described below produces active Rab GGTase by coinfection of Sf9 cells with viruses that encode for the ot and /3 subunits of the enzyme

Reagents

IPL-41 complete medium for monolayer and suspension culture of Sf9 cells: To prepare 500 ml, mix 450 ml IPL-41 medium (Sigma Chemi- cal Co.), 50 ml heat-inactivated (56 ° for 30 min) fetal calf serum (Sigma), 10 ml 50× yeastolate (GIBCO-BRL), 0.5 ml of 50 mg/

ml gentamicin (GIBCO-BRL), and 5 ml of 250/zg/ml Fungizone

20 D R O'Reilly, L K Miller, and V A Luckow, "Baculovirus Expression Vectors: A Laboratory Manual." Freeman, New York, 1992

[5] ENZYMES FOR PRENYLATION OF R a b PROTEINS 3 3 (GIBCO-BRL) For cells grown in suspension, add 10% Pluronic F-68 (GIBCO-BRL) to a final concentration of 0.1% (v/v)

2 x Grace's insect medium for plaque purification of recombinant bacu- loviruses: Dissolve 17.6 g Grace's insect medium ( J R H Biosciences) and 0.14 g NaHCO3 in 200 ml sterile water and adjust the pH to 6.2 with NaOH Filter through a 0.22-/.~m filter and add 8 ml of 50x yeastolate (GIBCO-BRL), 8 ml 50x lactalbumin hydrolyzate (GIBCO-BRL), 4 ml Fungizone, and 0.4 ml gentamicin at the con- centrations just given

2x Sea Plaque agarose for agarose overlay: Dissolve 1.5 g Sea Plaque agarose (FMC Bioproducts) in 40 ml water Autoclave for 45 min Neutral Red: Dissolve 100 mg Neutral Red (Sigma) in 20 ml water Filter through a 0.22-gm filter

5-Bromo-4-chloro-3-indoyl-/3-D-galactopyranoside (X-Gal) (Sigma): Prepare fresh as a 50-mg/ml solution in N, N-dimethylformamide

Construction and Production of Recombinant Baculovirus Encoding

ot and [3 Subunits o f Rab GGTase

The cDNAs that encode the a and [3 subunits of rat Rab GGTase are cloned into the EcoRI site of the baculovirus transfer vector, pVL1393

(Invitrogen), and the orientation is confirmed by restriction digestion The resulting plasmids, designated p V L - R a b G G T a and pVL-RabGGT[3,15 contain the sites necessary for homologous recombination into baculovi- ruses

Recombinant baculoviruses are produced by cotransfection of either pVL-RabGGTot or pVL-RabGGT[3 together with linearized BacPAK6 viral D N A (Clontech) by the lipofectin method} 1 Prior to transfection, Sf9 cells are seeded into plastic 6-well plates at 1 x 106 cells/well and are allowed to attach for 1 hr at 28 ° Two different transfection mixtures are prepared in polystyrene tubes by mixing i/zg of either pVL-RabGGTot or pVL-RabGGT[3, 2/xl linearized viral DNA, and 22/xl lipofectin (GIBCO- BRL) (diluted 2 parts lipofectin to i part sterile H20) and are incubated

at room temperature for 15 min Just before addition of the transfection mixtures, the cells are washed twice with 2 ml serum-free IPL-41 complete medium, and then 1 ml serum-free IPL-41 complete medium is added The transfection mixture (25/zl) is added, and the cells are incubated overnight

at 28 ° The next morning, the cells are refed with 2 ml of IPL-41 complete medium and incubation is continued for 2 days The medium is collected (designated "primary viral stock"), and the ceils are refed with 2 ml of

21 D R Groebe, A E (]hung, and C Ho, Nucleic Acids Res 18, 4033 (1990)

34 EXPRESSION, PURIFICATION, AND MODIFICATION [5] IPL-41 complete medium and incubated a further 72 hr After this period, the medium is collected (designated "secondary viral stock" and saved as

a backup reagent) At this point the cells can be checked for production

of the recombinant proteins by harvesting, detergent extraction, and immu- noblot analysis 14

A pure population of recombinant virus is produced by plaque purifica- tion of one viral clone followed by amplification For plaque purification, three 60-mm plates containing 1.2 × 106 Sf9 cells per plate are prepared, and the cells are allowed to attach for 1 hr at 28 ° Serial dilutions (10 -I,

10 -2, and 10 -3) of the primary viral stock in 2 ml of IPL-41 complete medium are added to the cells and incubated for 1 hr An agarose overlay solution

is prepared by mixing 25 ml of 2× Grace's medium, 5 ml heat-inactivated fetal calf serum, and 20 ml of 2× Sea Plaque agarose that has been auto- claved and cooled to 37 ° The medium is carefully aspirated, and 5 ml of the agarose overlay solution is gently poured over the cells with a plastic pipette The plates are incubated for 1 hr at room temperature so that the agarose solidifies and are then transferred to a 28 ° incubator for 4 days

On day 4, another sterile agarose solution containing 15 ml 2x Graces's insect medium, 3 ml fetal calf serum, 50/zg/ml Neutral Red, 250/xg/ml X-Gal, and 12 ml Sea Plaque agarose is prepared, overlaid (3 ml per plate)

so that the plate looks uniformly red, allowed to solidify, covered with aluminum foil, and incubated overnight at 28 ° The recombinant viruses will produce plaques that appear white, whereas plaques from nonrecombinant viruses will be blue A few white plaques are picked using a glass pipette and are then added directly (1 plaque/well) onto cells that have been seeded

in 6-well plates at 1 × 106 cells/well in 2 ml of IPL-41 complete medium The cells are incubated at 28 ° for 4 days, after which the medium is collected and designated "first amplification" medium For subsequent rounds of amplification, 0.5 ml of first amplification medium is added to a 50-ml suspension culture of Sf9 cells at 1 x 106 cells/ml and the cells are incubated for 4 days On day 4 the ceils are removed by centrifugation, and the supernatant, which contains the "amplified viral stock," is used for produc- tion of protein If desired, another plaque assay can be performed to deter- mine the viral titer

Production of Recombinant Rab GGTase in Sf9 Cells

Recombinant Rab GGTase is produced by coinfection of Sf9 cells with recombinant baculoviruses that encode the ot and /3 subunits A 50-ml suspension culture of Sf9 cells at 1 x 106 cells/ml growing at 28 ° in IPL-

41 complete medium plus 0.1% Pluronic F-68 is infected with the recombi- nant ~- and/3-encoding viruses at a multiplicity of infection of two o~ subunit

[5] ENZYMES FOR PRENYLATION OF Rab PROTEINS 35 and two/3 subunit viruses per cell This usually represents a 1 : 50 dilution

of the amplified viral stock as described earlier Forty-eight hours postinfec- tion, the cells are collected by centrifugation (2000 rpm, 10 min, room temperature), washed once in ice-cold phosphate-buffered saline, and re- pelleted, If necessary, the cell pellets are stored frozen at -70°; otherwise the cells are resuspended in 50 ml of lysis buffer containing 50 mM sodium HEPES (pH 7.2), 0.1 mM Nonidet P-40, (NP-40), 10 mM NaC1, 1 mM 2-mercaptoethanol (2-ME), 0.5 mM phenylmethylsulfonyl fluoride (PMSF),

5 t~g/ml pepstatin, 5/zg/ml leupeptin, and 5/xg/ml aprotinin and lysed by nitrogen cavitation in a Parr cell disruption bomb on ice for 15 min at 1000-1200 psi The lysate is then centrifuged at 100,000g (30,000 rpm in a Sorvall T647.5 rotor) for 30 min at 4 °, and the enzyme is purified from the supernatant as described below

Purification of R e c o m b i n a n t Rab GGTase from S f 9 Cytosol

Principle

High-level expression of recombinant Rab GGTase in Sf9 cells allows for a relatively simple purification procedure that consists of anion- exchange and gel filtration chromatography, which yields Rab GGTase with a purity of greater than 90% 15

Dialysis buffer: 20 mM sodium HEPES (pH 7.2), 0.1 mM NP-40, 10

mM NaCl, 1 mM DTT (or 1 mM 2-mercaptoethanol)

Q-Sepharose Chromatography

All steps are performed at 4 ° Twenty milliliters of Q-Sepharose high- performance resin (Pharmacia Biotech) is poured into a XK 16/10 column (Pharmacia) and connected to a FPLC system (Pharmacia) or a low pressure P-50 system (Pharmacia)

The column is initially washed with 60 ml of double-distilled water at

a flow rate of 2 ml/min and is then equilibrated with 30 ml of Q-Sepharose buffer A The Sf9 cell supernatant (50 ml) is loaded, and 8-ml fractions