small gtpases and their regulators, part d

Preface
In 1955 we edited three volumes of
Methods in Enzymology
(255, 256,
257) dedicated to small GTPases. Since then this field has exploded, and
these monomeric, regulatory proteins are now firmly established as a
common focus of interest in a wide variety of research areas including cell
and developmental biology, immunology, neurobiology, and, more re-
cently, microbiology. After talking with colleagues, it became apparent
that all three volumes needed to be significantly updated. We have, there-
fore, attempted to identify the major new areas and themes that have
emerged.
This volume covers the Rho GTPase family. These proteins are key
regulators of the actin cytoskeleton, and since the last volume on the subject
there has been significant progress in identifying and characterizing the
biochemical pathways associated with the three best characterized members
of this family, Rho, Rac, and Cdc42. In the past five years, interest has also
widened to a much broader community, as it has become clear that Rho
GTPases also participate in the regulation of many other signaling path-
ways, notably activation of the JNK and p38 MAP kinase pathways and
of transcription factors such as SRF and NF-KB. This ability to coordinately
regulate changes in the actin cytoskeleton with changes in gene transcription
and other associated activities appears to be conserved from yeast to
mammals.
When the last volumes were published, the large diversity of both down-
stream targets and upstream guanine nucleotide exchange factors that inter-
act with Rho GTPases was not fully appreciated. Not surprisingly, therefore,
these figure more prominantly this time around. Also, although it was
thought likely that Rho GTPases might participate in many processes de-
pendent on the organization of filamentous actin, it has now been directly
shown that these proteins control cell movement, phagocytosis, growth

cone guidance, and cytokinesis. An additional exciting new development
has been the identification and characterization of numerous proteins en-
coded by pathogenic bacteria that directly affect the activity of mammalian
Rho GTPases.
We very much hope that this and the accompanying volumes covering
the Ras family (Volumes 332 and 333) and the small GTPases involved in
membrane trafficking (Volume 329) will provide a useful source of practical
information for anyone entering the field. None of this would have been
XV
xvi PREFACE
possible without the talents and commitment of all our colleagues who
have contributed to these volumes. We are indebted to them.
ALAN HALL
WILLIAM E. BALCH
CHANNING J. DER
Contributors to Volume
325
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
KARON ABE (38),
Lineberger Comprehensive
Cancer Center, University of North Caro-
lina, Chapel Hill, North Carolina 27599
KLAUS AKTORIES (12),
Institut far Pharma-
kologie und Toxikologie, Albert-Ludwigs-
Universitiit Freiburg, D-79104 Freiburg,
Germany
MUTSUKI AMANO (14),
Division of Signal

Transduction, Nara Institute of Science and
Technology, Ikoma, Nara 630-0101, Japan
ANSER C. AZIM (22),
Division of Hematology,
Brigham and Women's, Hospital, Harvard
Medical School, Boston, Massachusetts
02115
DIANE L. BARBER (30),
Departments of Sto-
matology and Surgery, University of Cali-
fornia, San Francisco, California 94143
KURT L. BARKALOW (22, 31),
Division of He-
matology, Brigham and Women's Hospital
Harvard Medical School, Boston, Massa-
chusetts 02115
DAFNA BAR-SAGI (29),
Department of Molec-
ular Genetics and Microbiology, State Uni-
versi O, of New York, Stony Brook, New
York 11794-5222
GARY M. BOKOCH (28),
Departments" of Im-
munology and Cell Biology, The Scripps
Research Institute, La Jolla, California
92037
GIDEON BOLLAG (5, 6),
Onyx Pharmaceuti-
cals, Richmond, California 94806
DANIEL BROEK (4),

Department of Biochem-
istry and Molecular Biology, Keck School
()f Medicine, University of Southern Califor-
nia, Los Angeles, California 90033
SIIARON L. CAMPBELL (3),
Department of Bio-
chemistry and Biophysics. University of
North Carolina, Chapel Hill, North Caro-
lina 27599-7260
ix
EMMANUELLE CARON (41),
MRC Laboratory
for Molecular Cell Biology, University Col-
lege London, London WC1E 6BT, En-
gland, United Kingdom
CHRISTOPHER L. CARPENTER (18),
Division of
Signal Transduction, Beth Israel Deaconess
Medical Center, Boston, Massachusetts"
02215
FLAVIA CASTELLANO (25),
Institut Curie-
Recherche, CNRS UMR 144, 75248 Paris
Cedex 05, France
CHESTER E. CHAMBERLAIN (35),
Department
of Cell Biology, The Scripps Research Insti-
tute, La Jolla, California 92037
PHILIPPE CHAVRIER
(25),

lnstitut Curie-Re-
cherche, CNRS UMR 144, 75248 Paris
Cedex 05, France
EDWIN CHOY (10),
Department of Medicine,
Massachusetts" General Hospital, Boston,
Massachusetts 02114
JOHN G. COLLARD (26, 36),
Division of Cell
Biology, The Netherlands" Cancer Institute,
1066 CX Amsterdam, The Netherlands
ANNE M. CROMPTON (5),
Onyx Pharmaceuti-
cals, Richmond, California 94806
GIOVANNA M. D'ABACO (37),
Cancer Re-
search Campaign for Cell and Molecular
Biology, Chester Beatty Laboratories, Insti-
tute of Cancer Research, London S W3 6JB,
England, United Kingdom
BALAKA DAS (4),
Department of Biochemis-
try and Molecular Biology, Keck School of
Medicine, University of Southern Califor-
nia, Los Angeles, California 90033
SHERYL P. DENKER (30),
Department of Sto-
matology, University of California, San
Francisco, California 94143
X

CONTRIBUTORS TO VOLUME
325
CHANNING J. DER (38),
Lineberger Compre-
hensive Cancer Center, The University of
North Carolina, Chapel Hill, North Caro-
lina 27599
JOHN F. ECCLESTON (7),
Division of Physical
Biochemistry, National Institute for Medical
Research, London NW7 1AA, England,
United Kingdom
EVA E. EVERS (36),
Division of Cell Biology,
The Netherlands Cancer Institute, 1066 CX
Amsterdam, The Netherlands
TOREN FINKEL (27),
National Heart, Lung,
and Blood Institute, Laboratory of Molec-
ular Biology, National Institutes of Health,
Bethesda, Maryland 20892-1650
ALYSON E. FOURNIER (42),
Department of
Neurology, Yale University School of Medi-
cine, New Haven, Connecticut 06520
ANDREA FRIEBEL (8),
Max von Pettenkofer-
Institut, Ludwig Maximilians Universitiit,
80336 Munich, Germany
MICHAEL A. FROHMAN (17),

Department of
Pharmacology and Institute for Cell and
Developmental Biology, State University of
New York, Stony Brook, New York
11794-8651
YtXIN Fu (44),
Section of Microbial Pathogen-
esis, Boyer Center for Molecular Medicine,
Yale University School of Medicine, New
Haven, Connecticut 06536-0812
KEIGI FUJIWARA (33),
Department of Struc-
tural Analysis, National Cardiovascular
Center Research Institute, Osaka 565-
8565, Japan
YUKO FUKATA (14),
Department of Cell Phar-
macology, Nagoya University School of
Medicine, Nagoya AICHI 466-8550, Japan
JORGE E. (}ALAN (44),
Section of Microbial
Pathogenesis, Boyer Center for Molecular
Medicine, Yale University School of Medi-
cine, New Haven, Connecticut 06536-0812
PETER GIERSCHIK (16),
Department of Phar-
macology and Toxicology, University of
Ulm, D-89081 Ulm, Germany
GASTON G. M. HABETS
(5),

Onyx Pharmaceu-
ticals, Richmond, California 94806
KLAUS M. HAHN (35),
Department of Cell
Biology, The Scripps Research Institute, La
Jolla, California 92037
ALAN HALL (41),
MRC Laboratory for Mo-
lecular Cell Biology, University College
London, London WCIE 6BT, England,
United Kingdom
ANDREW D. HAMILTON (34),
Department of
Chemistry, Yale University, New Haven,
Connecticut 06511
JAEWON HAN (4),
Department of Vascular Bi-
ology, The Scripps Research Institute, La
JoUa, California 92037
WOLF-DIETRICH HARDY (8),
Max von Pet-
tenkofer-lnstitut, Ludwig Maximilians Uni-
versiti#, 80336 Munich, Germany
MATTHEW J. HART (6),
Onyx Pharmaceuti-
cals, Richmond, California 94806
JOHN H. HARTWIG (22, 31),
Division of Hema-
tology, Brigham and Women's Hospital,
Harvard Medical School, Boston, Massa-

chusetts 02115
PATRICK HEARING (29),
Department of Mo-
lecular Genetics and Microbiology, State
University of New York, Stony Brook, New
York 11794-5222
MARK R. HOLT (32),
Physiology Department,
University College London, London WC1E
6J J, England, United Kingdom
JON P. HUTCHINSON (7),
Division of Physical
Biochemistry, National Institute for Medical
Research, London NW7 1AA, England,
United Kingdom
DARIA ILLENBEROER (16),
Department of
Pharmacology and Toxicology, University
of Ulm, D-89081 Ulm, Germany
TOSHIMASA ISHIZAKI (24),
Department of
Pharmacology, Kyoto University Faculty of
Medicine, Kyoto 606-8315, Japan
LENNERT JANSSEN (26),
Division of Cell Biol-
ogy, The Netherlands Cancer Institute, 1066
CX Amsterdam, The Netherlands
DANIEL G. JAY (43),
Department of Physiol-
ogy, Tufts University School of Medicine,

Boston, Massachusetts 02111
CONTRIBUTORS TO VOLUME 325 xi
GARETH E. JONES (40),
Randall Centre for
Molecular Mechanisms of Cell Fanction,
King's College London, London SE1 1UL,
England, United Kingdom
Kozo KAIBUCHI (14),
Department of Cell
Pharmacology, Nagoya University School
of Medicine, Nagoya AICHI 466-8550, Ja-
pan and Division of Signal Transduction,
Nara Institute of Science and Technology,
lkoma, Nara 630-0101, Japan
ROBERT G. KALB (42),
Department of Neurol-
ogy, Yale University School qf Medicine,
.New Haven, Connecticut 06520
YASUNORI KANAItO (17),
Department of
Pharmacology, Tokyo Metropolitan Msti-
tute of Medical Science, Tokyo 113-8613,
Japan
YUMJKO KANO (33),
Department of Structural
Analysis, National Cardiovascular Center
Research Institute, Osaka 565-8565, Japan
KAzuo KATOH (33),
Department of Structural
Analysis, National Cardiovascular Center

Research Institute, Osaka 565-8565, Japan
CHARLES C. KING (15, 28),
Department of Im-
munology, The Scripps Research Institute,
La .lol&, Cal(fornia 92037
UEEA G. KNAUS (15),
Department of Immu-
nology, The Scripps Research Institute, La
Jolla, California 92037
ANNA KOFFER (32),
Physiology Department,
University College London, London WC1E
6J J, England, United Kingdom
VADIM S. KRAYNOV (35),
Department of Cell,
Biology, The Scripps Research Institute, La
Jolla, Califi)rnia 92037
IAN O. MACARA (1),
The Markey Center fi)r
Cell Signaling, University of Virginia, Char-
lottesville, Virginia 22908
LAURA M. MACHESKY (20),
Division of Mo-
lecular Cell Biology. School of Biosciences,
University of Birmingham, Birmingham
B15 2TT, England, United Kingdom
AKIKO MAMMOTO (9),
Department of Molec-
ular Biology and Biochemistry, Osaka Uni-
versity Graduate School of Medicine, Fac-

uhv of Medicine, Osaka 565-0871, Japan
DANNY MANOR (13),
Division o¢" Nutritional
Sciences, Cornell University, Ithaca, New
York 14853
FRITS MICHIELS (26),
Galapagos Genomics,
2333 AL Leiden, The Netherlands
MICttAEL MOOS (11),
lnstitutfiir Medizinische
Mikrobiologie, Universitiit Mainz, D-55101
Mainz, Germany
ANDREW J. MORRIS (17),
Department of Phar-
macology and institute for Cell and Devel-
opmental Biology, State University of New
York, Stony Brook, New York 11794-8651
RAYMOND MOSTELLER (4),
Department of
Biochemistry and Molecular Biology, Keck
School of Medicine, University qf Southern
Cal(fi~rnia, Los Angeles, California 90033
R. DYCHE MULLINS (20),
Department of' Cel-
hdar and Molecular Pharmacology, Univer-
sity of California School of Medicine, San
Francisco, California 94143
ROBERT K. NAKAMOIO (2),
Department of
Molecular Physiology and Biological Phys-

ics, University of Virginia, Charh)ttesvilh',
Virginia 22908-07.36
SHUH NAI~.UMIYA (24).
Department of Phar-
macology, Kyoto Univers'ity Faculty of
Medicine, Kyoto 606-8.315, Japan
CIIERYL L. NEUDAUER (1)~
The Markey Cen-
ter for Cell Signaling, University of Virginia,
Charlottesville, Virginia 22908
MARGARE l'A NIKOLIC (19),
Molectdar Neuro-
biology Group, King's College, London,
Enghmd, United Kingdom
CATHERINE D. NOBES (39),
MRC Laboratory
for Molecular Cell Biology and Department
of Anatomy and Developmental Biology,
University College London, London WCI E
6BT, England, United Kingdom
GARRY NOLAN (26),
Stanford University
School of Medicine, Stanford, California
94305
MICHAEL F. OLSON (37),
Cancer Research
Campaign for Cell and Molecular Bioh)gy,
Chester Beatty Laboratories, Institute of
Cancer Research, London SW3 6JB, En-
gland, United Kingdom

xii
CONTRIBUTORS TO VOLUME 325
JAYESI-I C. PATEL (41),
MRC Laboratory for
Molecular Cell Biology, University College
London, London WC1E 6BT, England,
United Kingdom
DANIELLE PEVERLY-MITCHELL (5),
Onyx
Pharmaceuticals, Richmond, California
94806
MARK PHILIPS (10),
Departments of Medicine
and Cell Biology, New York University
School of Medicine, New York, New
York 10016
PAUL W. READ (2),
Department of Molecu-
lar Physiology and Biological Physics, Uni-
versity of Virginia, Charlottesville, Virginia
22908-0736
ABINA M. REILLY (15),
Department oflmmu-
nology, The Scripps Research Institute, La
Jolla, California 92037
XIANG-DONG REN (23),
State University of
New York, Stony Brook, New York
11794-8165
ANNE J. RIDLEY (40),

Ludwig Institute for
Cancer Research, London W1P 8BT, En-
gland, United Kingdom
KATRIN RITI~INGER (7),
Division of Protein
Structure, National Institute for Medical
Research, London NW7 1AA, England,
United Kingdom
WILLIAM ROSCOE (6),
Onyx Pharmaceuticals,
Richmond, California 94806
KENT L. ROSSMAN (3),
Department of Bio-
chemistry and Biophysics, University of
North Carolina, Chapel Hill, North Caro-
lina 27599-7260
LURAYNNE C. SANDERS (28),
Department of
Immunology, The Scripps Research Insti-
tute, La Jolla, California 92037
TAKUYA SASAKI (9),
Department of Biochem-
istry, The University of Tokushima, School
of Medicine, Kuramoto, Japan
GUDULA SCHMIDT (12),
Institut far Pharma-
kologie und Toxikologie, Albert-Ludwigs-
Universitiit Freiburg, D-79104 Freiburg,
Germany
FRIEDER SCHWALD (16),

Department of Phar-
macology and Toxicology, University of
Ulm, D-89081 Ulm, Germany
MARTIN ALEXANDER SCHWARTZ (23),
The
Scripps Research Institute, La Jolla, Califor-
nia 92037
SA'I'D M. SEBTI (34),
Drug Discovery Program,
H. Lee Moffitt Cancer Center and Research
Institute, University of South Florida,
Tampa, Florida 33612
HIROAKI SHIMOKAWA (14),
Research Institute
of Angiocardiology and Cardiovascular
Clinic, Kyushu University School of Medi-
cine, Fukuoka 812-8582, Japan
PATRICIA A. SOLSKI (38),
Lineberger Compre-
hensive Cancer Center, University of North
Carolina, Chapel Hill, North Carolina
27599
ILONA STEPHAN (16),
Department of Pharma-
cology and Toxicology, University of Ulm,
D-89081 Ulm, Germany
STEPHEN M. STRITrMATrER (42),
Department
of Neurology, Yale University School of
Medicine, New Haven, Connecticut 06520

DANIEL M. SULLIVAN (27),
National Heart,
Lung, and Blood Institute, Laboratory of
Molecular Biology, National Institutes of
Health, Bethesda, Maryland 20892-1650
MARC SYMONS (5),
Onyx Pharmaceuticals,
Richmond, California 94806
KAZUO TAKAHASHI (9),
Second Department
of Internal Medicine, Chiba University
Medical School, Chiba 260-0856, Japan
YOSHIMI TAKAI (9),
Department of Molecular
Biology and Biochemistry, Osaka Univer-
sity Graduate School of Medicine~Faculty
of Medicine, Osaka 565-0871, Japan
LAURA J. TAYLOR (29),
Department of Molec-
ular Genetics and Microbiology, State Uni-
versity of New York, Stony Brook, New
York 11794-5222
JEAN P. TEN KLOOSTER (36),
Division of Cell
Biology, The Netherlands Cancer Institute,
1066 CX Amsterdam, The Netherlands
KIMBERLEY TOLIAS (18),
Division of Signal
Transduction, Beth Israel Deaconess Medi-
cal Center, Boston, Massachusetts 02215

CONTRIBUTORS TO VOLUME
325
Xlll
LI-HUEI TSAI (19),
Howard Hughes Medical
Institute, Department of Pathology, Har-
vard Medical School, Boston, Massachu-
setts 02115
MASAYOSHI UEHATA (24),
Drug Discovery
Laboratories, WelFide (Yoshitomi) Corpo-
ration, Osaka 573-1153, Japan
RoB A. VAN DER KAMMEN (26, 36),
Division
of Cell Biology, The Netherlands' Cancer
Institute, 1066 CX Amsterdam, The Nether-
lands'
CHRISTOPH VON EICHEL-STREIBER
(11),
Ver-
fligungsgebziude fiir Forschung und Ent-
wicklung, lnstitut far Medizinisch Mikrobi-
ologie und Hygiene, Johannes Gutenberg-
Universitiit, 55101 Mainz, Germany
AMY B. WALSH (29),
Department of Molecu-
lar Genetics and Microbiology, State Uni-
versity of New York, Stony Brook, New
York 11794-5222
ERIC V. WONG (43),

Department of Physiol-
ogy, Tufts University School of Medicine,
Boston, Massachusetts 02111
WEIHONG YAN (30),
Department of Stoma-
tology, University of California, San Fran-
cisco, California 94143
YUE ZHANG (17),
Department of Pharmacol-
ogy and Institute for Cell and Develop-
mental Biology, State University of New
York, Stony Brook, New York 11794-8651
DANIEL ZICHA (40),
Imperial Cancer Re-
search Fund, London WC2A 3PX, En-
gland, United Kingdom
SALLY H. ZIGMOND (21),
Biology Depart-
ment, University of Pennsylvania, Philadel-
phia, Pennsylvania 19104-6018
[ 1 ] CHARACTERIZATION OF TC10 3
[1] Purification and Biochemical Characterization
of TC 1 0
By CHERYL L. NEUDAUER and IAN G. MACARA
Introduction
TC10 is a member of the Rho family of small GTPases. We have
previously characterized the biochemistry, cellular effects, and effector in-
teractions of TC10.1 This study established TC10 as a distinct member of
the Rho family most closely related to Cdc42. In NIH 3T3 cells, the ectopic
expression of hemagglutinin (HA)-tagged, gain-of-function TC10 induces

long filopodia and loss of stress fibers. TC10 interacts with a subset of those
effector proteins that bind to Cdc42. TC10 also interacts with several distinct
proteins that are specific for TC10. 2 This article describes the methods used
for the mutagenesis of TC10 and the set of vectors used for the purification
of recombinant proteins and mammalian expression of TC10. It also de-
scribes the biochemical characterization of TC10 and various methods used
to study the interaction of TC10 with putative effector proteins.
Mutagenesis and Subcloning of TC10
We have tested several methods for the introduction of point mutations
and have found megaprimer polymerase chain reaction (PCR) to be a
relatively consistent, cost effective, and reliable technique. 3 Briefly, an inter-
nal primer is designed that contains the nucleotide substitution(s). An initial
round of PCR is performed with this primer and a primer to either the 5'
or 3' end of the sequence. In general, we include a
BamHI
site in the 5'
primer and an
EcoRI
site in the 3' primer to facilitate subcloning. Restric-
tion enzymes usually cut the ends of PCR products inefficiently and are
therefore digested with high concentrations of
BamHI
and
EcoRI
at 37 °
for greater than 4 hr.
To facilitate the expression of TCI0 and other proteins in bacteria,
yeast, and mammalian cells, we have designed a set of vectors with similar
I C. L. Neudauer, G. Joberty, N. Tatsis, and I. G. Macara, Curr. Biol. 8, 1151 (1998).
2 G. Joberty, unpublished results (1999).

3 S. Batik, in "'PCR Protocols: Current Methods and Applications" (B. A. White, ed.),
p. 277. Humana Press, Totowa, NJ, 1993.
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
MET1 tODS IN ENZYMOLOGY, VOL. 325 0076-6879/00 $30.00
4
PURIFICATION, MODIFICATION, AND REGULATION
[ 1]
cloning sites (Table I). The majority of these vectors produce N-terminally
tagged fusion proteins; C-terminal tagging of the small GTPases is usually
avoided as most of these proteins undergo posttranslational modification
(e.g., prenylation, carboxymethylation) at their C termini. Each vector
contains a BamHI site in the same reading frame as pGEX-2T (Amersham
Pharmacia, Piscataway, N J; Fig. 1).
The pK series of vectors derives expression from a cytomegalovirus
(CMV) promoter and contains splice donor and acceptor sites upstream
of the initiation codon to increase the efficiency of mRNA export from the
nucleus. The vectors contain a simian virus 40 (SV40) origin, so they will
replicate in COS-7 cells (which contain the SV40 large T antigen). They
are designed for high-level expression in transient transfections and do not
contain a eukaryotic selectable marker. This set of vectors allows for the
rapid characterization of TC10 or other proteins by prokaryotic expression
and purification and by mammalian expression and immunoprecipitation,
immunoblotting, or immunofluorescence. The purification methods are
listed in Table I. The antibodies used and their concentrations for immu-
noblots or immunofluorescence are listed in Table II.
TABLE
I
VECTOR SUMMARY
Parent

vector Vector Tag Purification Expression
pQE70 pQNzz ZZ lgG Sepharose Prokaryotic
Oiagen
pRK7 Mammalian
pRK7 pKH3 Triple HA 12CA5 with Sigma protein Mammalian
A-Sepharose
pRK7 pKMyc Myc 9El0 with Amersham Phar- Mammalian
macia GammaBind Plus
Sepharose
pRK7 pKFLAG FLAG Sigma anti-FLAG Mammalian
M2-agarose
pRK7 pRK7-GFP GFP Santa Cruz anti-GFP with Mammalian
Sigma protein
A Sepharose
pRK7 pKNzz ZZ IgG Sepharose Mammalian
pGBT9 pGBT10 GAL4 DNA-bind- Yeast
Clontech ing domain
pVP16 pVP16-CP GAL4 activation Yeast
domain
[ 1] CHARACTERIZATION OF TC10
pGEX-2T
Smal
pQNzz
NcoI
r "'D B amHI NotI EcoRI BgllI
CC ATG~ GCJG 'CGG CCG C~A ATT C~G ATC T I
pRK7
HindllI PstI SalI XbaI BamHI EcoRI ClaI
~'CTG CAG"GTC GAC'~FCT AGA'~GA TcciccG GG~'~'~'IA~
PKH3

BamHI EcoRI ClaI
iGGA TCCUGAA TFCtAtAT CGA T i
PKMyc
Sinai
iNheI Notl EcoRI ClaI
CGG ~3CT AGC t GG~3 CGG CCG C~lqGAA TTCnATC GAT i
pKFLAG
BamHI EeoRI ClaI Sill
tGGA TCC~tGAA TI"C~AtAT CGA qtGG CCG CCA TGG CC~
PRKT-GFP
BamHI EcoRI ClaI
~~GA GAA ~TC
pKNzz
BamHI Notl EeoRI ClaI
~GCIG CGG CCG CbA ATT C~A~
pGBT10
BamHI AatI1 EcoRI
iGGA TCCIIGAC GTCI~
VP16-CP
BamHI AatlI EcoRI
IGGA TCCJIGAC GTCtIGGT ACC I
FIG. 1. Multiple cloning sites of expression vector set. These vectors were designed to
placc the
BamHI
cloning site in the same reading frame as pGEX-2T (Amersham Pharmacia,
shown as reference).
6
PURIFICATION, MODIFICATION, AND REGULATION [ l]
TABLE II
ANTIBODY DILUTIONS FOR IMMUNOBLOTFING AND IMMUNOFLUORESCENCE

Immunoblotting Immunofluorescenc(
concentration concentration
Antibody" Company (/xg/ml) (b~g/ml)
Anti-GST Santa Cruz (Santa Cruz, 0.008 0.2
CA)
0.2 3.75
BAbCO (Richmond, CA) 1 2
0.4 4
Kodak (Rochester, NY) 0.66 10
Molecular Probes 2 4
(Eugene, OR)
12CA5 (anti-HA)
Polyclonal anti-HA
9El0 (anti-myc)
Anti-FLAG M2
Polyclonal anti-GFP
a All antibodies are monoclonal, except those indicated.
Purification of Glutathione S-Transferase (GST)-TC10
Fusion Proteins
To decrease the loss of plasmid due to destruction of the ampicillin by
the ~-lactamase product of the ampicillin resistance gene, grow overnight
cultures as lawns on four LB/ampicillin plates. Scrape the colonies off the
plates into 1 liter of LB/ampicillin and grow at either room temperature
or 37 ° with shaking until the OD600 = 0.8. Induce the cultures with 1 mM
isopropyl-/3-D-thiogalactoside (IPTG) at room temperature for 2-4 hr with
shaking. Resuspend the pelleted cells in a lysis buffer containing MgCI2
[50 mM Tris, pH 8, 1 mM MgCI2, 0.1 mM EDTA, 1 mM dithiothreitol
(DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 25/,~g/ml leupeptin,
10/xg/ml DNase I, and 1 mg/ml lysozyme]. MgC12 is necessary to maintain
guanine nucleotide complexed to the TC10 proteins. In the absence of a

complexed nucleotide, small GTPases rapidly denature. In general, we
have found lysis in a French press to provide a higher fraction of soluble,
functional protein than sonication and/or freeze-thawing. Certain point
mutations in TC10 (e.g., Q75L) substantially reduce the solubility of the
GST-fusion protein, particularly when the cells are lysed by sonication.
To prepare recombinant TC10 lacking the N-terminal GST tag, either
cleave directly from the glutathione-Sepharose beads or in solution after
elution from the beads. To cleave from the beads, wash the beads with
thrombin cleavage buffer (50 mM Tris, pH 7.5, 150 mM NaC1, 2.5 mM
CaC12) and incubate with thrombin at 4 ° overnight. Remove the thrombin
by incubation with p-aminobenzamide-Sepharose (Sigma, St. Louis, MO;
washed first with thrombin cleavage buffer) at 4 ° for 30 min. To cleave in
solution, first remove the glutathione by passage over a PD10 column
(Amersham Pharmacia, Piscataway, N J) or a Centricep spin column
[ 11 CHARACTERIZATION OF TC10 7
(Princeton Separations, Adelphia, N J), with buffer exchange into thrombin
cleavage buffer. Remove the GST by incubation with glutathione-
Sepharose at 4 ° for 30 min and then remove the thrombin with washed p-
aminobenzamide-Sepharose. Concentrate the proteins and exchange into
appropriate buffers using a Centricon-30 or Centricon-10 (Millipore, Bed-
ford, MA). Freeze the proteins prepared in this manner in liquid nitrogen
and store at -80 ° , under which conditions they are stable for several months.
Conversion of [a-32p]GTP to [c~-32p]GDP
Reagents
100 mM Magnesium acetate
1 mg/ml Nucleotide diphosphate kinase (NDPK; Sigma)
100 mM Uridine diphosphate (UDP)
1 M HEPES, pH 7.4
25 mM DTT
10 mM EDTA, pH 7.0

Glycerol
Distilled water
[o~-32p]GTP (3000-5000 Ci/mmol)
1 N NaOH
1 N
HC1
Procedure
In a microcentrifuge tube, combine 4 /xl magnesium acetate, 1.5 txl
NDPK, 4 tzl UDP, 4 txl HEPES, 2 ixl DTT, 2/xl EDTA, 40/xl glycerol, 40
/xl distilled water, and 40/xl [a-32p]GTP and incubate at 30 ° for 30 minutes. 4
Stop the reaction by the addition of 8/xl 1 N NaOH and incubate on ice
for 10 min. Neutralize the reaction by the addition of 8/xl HCI. Aliquot
and store at -20 °. Detect the conversion efficiency by thin-layer chromatog-
raphy on polyethyleneimine-cellulose plates (Baker, Phillipsburg, NJ) with
0.75 M Tris base, 0.5 M LiC1, and 0.45 M HC1 running buffer. 5 Soak the
plates in methanol prior to and after use to remove buffer.
Loading Recombinant TC 10 with Labeled Nucleotide
Small GTPases are loaded with radiolabeled nucleotide in the presence
of EDTA to chelate Mg 2+ ions. After loading, the nucleotide is trapped
on the protein by the addition of excess Mg2+. 6
4 I. G. Macara and W. H. Brondyk,
Methods Enzymol.
257, 117 (1995).
B. R. Bochner and B. N. Ames,
J. Biol, Chem.
257, 9759 (1982).
6E. S. Burstein and I. G. Macara,
Biochem. J.
282, 387 (1992).
8 PURIFICATION, MODIFICATION, AND REGULATION [1]

Procedure
In a microcentrifuge tube, combine 1-5/zg of recombinant TC10, 5/xl
1% (w/v) bovine serum albumin (BSA), 1 /xl [ce-32p]GTP or [y-32p]GTP
(3000-5000 Ci/mmol) or 3.8/M [a-3ep]GDP (equivalent to 1 tzl GTP), and
25 mM MOPS, pH 7.1, and 1 mM EDTA to 50/zl and incubate on ice for
20 min. Add 1/xl 1 M MgC12 and incubate on ice for an additional 10 rain.
Store loaded proteins on ice prior to use.
To quantitate the amount of complexed nucleotide, bind loaded TC10
to nitrocellulose filters (Millipore, Bedford, MA; HAWP02400) in the
presence of quench buffer (15 mM sodium phosphate, 10 mM MgC12,
1 mM ATP). Wash filters twice with quench buffer. Measure the radioactiv-
ity bound to the filters by scintillation counting. To remove unincorporated
nucleotide, pass loaded TC10 over a PD-10 or Centricep column, equili-
brated in appropriate buffer (containing ->1 mM MgC12).
Biochemical Characterization of TC 10
To determine the intrinsic GTPase and exchange activities of TC10, 7'~
load recombinant protein with [y-32p]GTP for GTPase activity or [o~-
32p]GTP for exchange activity as described previously. Dilute loaded TC10
in 25 mM MOPS, pH 7.1, 1 mM GTP, I mM GDP, 5 mM MgC12, and
incubate at 30 °. Remove aliquots at timed intervals, filter bind as described
previously, and quantitate by scintillation counting. The koff and k~at values
are calculated assuming single-exponential kinetics. However, the rate of
loss of [T-32p]GTP from the TC10 is actually the sum of the release and
hydrolysis rates. Therefore, it is necessary to correct the apparent kcat value
by subtraction of ko,.
To determine whether a GTPase-activating protein (GAP) has activity
on TC10, load recombinant, cleaved TC10 with [y-32p]GTP as described
earlier. Serially dilute the GAP protein in an appropriate buffer in threefold
steps. In a microcentrifuge tube, combine 452.5/xl 25 mM MOPS, pH 7.1,
5 kd 100 mM GTP, 5/xl 100 mMGDP, and 2.5/zl 1 M MgC12 and incubate

at 30 °. Add 25/zl of diluted GAP or buffer and incubate at 30 °. Initiate
the reaction by the addition of 10 tzl [y-32p]GTP-TC10, vortex, and incubate
at 30 ° for 3 min. At to and at various time points, remove 20 tzl for filter
binding and quantitate by scintillation counting. Intrinsic kcat values are
calculated and subtracted from k~at values in the presence of GAP, assuming
single-exponential kinetics. The apparent affinity of GAP for TC10 is esti-
7 j. B. Gibbs, M. D. Schaber, W. J. Allard, I. S. Sigal, and E. M. Scolnick,
Proc. Natl. Acad.
Sci. U.S.A.
85, 5026 (1988).
8 j. John, M. Frech, and A. Wittinghofer,
J. BioL Chem.
263, 11792 (1988).
[ 1] CHARACTERIZATION OF TC10 9
mated as the GAP concentration yielding a half-maximal acceleration of hy-
drolysis.
Kinase Assays
Racl and Cdc42 have been shown to activate Jun N-terminal kinase
(JNK)9 12 and p21-activated kinase (PAK)J 3 To determine if TC10 and its
effectors can activate these kinases, tagged TC10, effector proteins, and
kinase are coexpressed by transient transfection, immunoprecipitated, and
assayed
in vitro.
In some cases, coexpression of a kinase with another
protein may diminish the expression of one of the proteins. In these cases,
the amount of plasmid transfected is modified to obtain similar expression
levels or the tagged kinase is immunoprecipitated and activated
in vitro
with recombinant TC10 and/or effector proteins.
JNK assays are performed similar to Derijard

et al.14
and Coso
et al. 9
Cotransfect pKH3-JNK with pKMyc-TC10(Q75L) into NIH 3T3 or
COS-7 cells. To determine the basal activation of JNK, transfect one plate
of cells with pKH3-JNK (and empty vector to normalize plasmid levels).
To test the effect of putative TC10 effectors on JNK activity, cotransfect
the effector in pKMyc with pKH3-JNK or with pKH3-JNK and pKMyc-
TC10(Q75L). At 24 hr after transfection, transfer cells to serum-free me-
dium and starve overnight. Place cells on ice, wash once with phosphate-
buffered saline (PBS), and lyse cells with 400 /xl of lysis buffer [25 mM
HEPES, pH 7.4, 0.3 M NaC1, 1.5 mM MgC12, 0.5 mM DTT, 20 mM/3-
glycerophosphate, 1 mM sodium vanadate, 1 /xM okadaic acid, 20/,g/ml
aprotinin, 10 txg/ml leupeptin, 1 mM PMSF, and 0.1% (v/v) Triton X-100].
Scrape the cells from the plate and centrifuge at 13,000g at 4 ° for 5 min.
Remove 50 /xl of each soluble lysate to determine protein expression by
immunoblotting. Immunoprecipitate HA-tagged JNK from the soluble su-
pernatant with 3/,g 12CA5 at 4 ° for 1 hr, followed by incubation with 30
/,1 of protein A-Sepharose (washed with lysis buffer) at 4 ° for 1 hr. Wash
the beads three times with 2 mM sodium vanadate, 1% Igepal in PBS, once
with 0.1 M MOPS, pH 7.5, 0.5 M LiC1, and once with kinase buffer (12.5
mM MOPS, pH 7.5, 12.5 mM/3-glycerophosphate, 7.5 mM MgC12, 0.5 mM
~ O. A. Coso, M. Chiariello, J. C. Yu, H. Teramoto, P. Crespo, N. Xu, T. Miki. and J. S.
Gutkind,
Cell
81, 1137 (1995).
m S. Bagrodia, B. Derijard, R. J, Davis, and R. A. Cerione,
J. Biol. Chem.
270, 27995 (1995).
~ A. Minden, A. Lin, F. X. Claret. A. Abo, and M. Karin,

Cell
81, 1147 (1995).
12 M. F. Olson, A. Ashworth, and A. Hall,
Science
269, 1270 (1995).
13 E. Manser, T. Leung, H. Salihuddin, Z. S. Zhao, and k. Lim,
Nature
367, 40 (1994).
~a B. Derijard, M. Hibi, I. H. Wu, T. Barrett, B. Su, T. Deng, M. Karin, and R. J. Davis,
Cell
76, 1025 (1994).
10 PURIFICATION, MODIFICATION, AND REGULATION [1]
EGTA, 0.5 mM NaF, and 0.5 mM sodium vanadate). Resuspend the beads
in 300/xl kinase buffer; remove 30 tzl of resuspended beads to analyze the
amount of immunoprecipitated protein by immunoblotting. Pellet beads
and initiate JNK reactions by the addition of 30/xl kinase buffer containing
2/xg recombinant GST-Jun(1-79) and 2 ~Ci [T-32p]ATP (6000 Ci/mmol).
Incubate reactions at 30 ° for 20 min and terminate by the addition of 10
/xl 4;4 SDS-PAGE sample buffer. Fractionate phosphorylated substrates
with 12% SDS-PAGE and visualize by fluorography. Fractionate expressed
and immunoprecipitated proteins with 12% SDS-PAGE and transfer to
nitrocellulose for immunoblotting. To avoid detection of the antibody used
in the immunoprecipitation by the anti-mouse secondary antibody, use
12CA5 or 9El0 coupled directly to horseradish peroxidase.
Our methods to assay the activation of PAK are based on those of
Knaus
et aL 15
and Lamarche
et aL ]6
Because aPAK expression is often

perturbed by its cotransfection with other plasmids, pCMV6M-aPAK (a
gift from G. Bokoch, Scripps Research Institute, La Jolla, CA) is transfected
alone into NIH 3T3 or COS-7 cells. PAK is immunoprecipitated as de-
scribed earlier with 1/zg polyclonal anti-PAK antibody (Santa Cruz Bio-
technology, Inc., Santa Cruz, CA) and protein A-Sepharose. Alternatively,
Myc-aPAK can be immunoprecipitated with 4/zg 9El0 and GammaBind
Plus Sepharose. Stimulate the immunoprecipitated
aPAK
by the addition
of 45 tzl recombinant TC10, loaded as described earlier in the presence of
2 mM guanylyl imidodiphosphate tetralithium salt (GMP-PNP; Boehringer
Mannheim, Indianapolis, IN), and incubate on ice for 5 min. Initiate PAK
reactions by the addition of 30 tzl kinase buffer containing 5 /zg of the
substrate, myelin basic protein (Sigma, St. Louis, MO), and 5 tzCi
[y-32P]ATP. Incubate reactions at 30 ° for 20 min. Terminate reactions by
the addition of 30/xl 4;4 SDS-PAGE sample buffer and analyze results
as described earlier.
Interaction of TC 10 with Putative Effectors
We routinely use five assays to detect the interaction of TC10 with
putative effectors. These assays include yeast two-hybrid interactions, over-
lay assays, coprecipitation assays, coimmunoprecipitation assays, and
in
vitro
competition assays. The yeast two-hybrid assay is the most sensitive
of these, but it does not determine if the interaction is direct and yields
little information about affinity. An interaction can appear to be much
higher affinity in the yeast two-hybrid interaction than
in vitro
due to self-
15 U. G. Knaus, S. Morris, H. J. Dong, J. Chernoff, and G. M. Bokoch,

Science
269, 221 (1995).
16 N. Lamarche, N. Tapon, L. Stowers, P. D. Burbelo, P. Aspenstrom, T. Bridges, J. Chant,
and A. Hall,
Cell
87, 519 (1996).
[ 1] CHARACTERIZATION OF TC10 11
activation by the effector. The yeast two-hybrid interactions have been
described elsewhere 17 and will not be discussed here. Coimmunoprecipita-
tion also does not necessarily detect a direct interaction. Overlay and co-
precipitation assays measure direct interactions but require nanomolar af-
finities. The in vitro competition assay is the most sensitive. It measures
direct interactions, and we have determined affinities with KD values of
approximately 20/xM.
Overlay Assays
In the overlay assay, a putative effector protein is immobilized on nitro-
cellulose and is then overlaid with TC10 that has been complexed with
radioactive nucleotide. The guanine nucleotide specificity can be examined
by loading recombinant TC10 with either [o~-32P]GTP or [o~-32p]GDP and
overlaying two filters bound to the same putative effector proteins. The
specificity of the interaction of the effector with small GTPases can be
assessed by overlaying individual filters with various GTPases. To decrease
the likelihood of false positives due to the dimerization of GST, it is impor-
tant to avoid using GST-fusions of both the effector and the GTPase. Our
methods for the overlay assay are modified from Manser et al.18
Procedure.
Fractionate recombinant proteins or lysates of cells express-
ing effector proteins by SDS-PAGE followed by transfer of the proteins
to nitrocellulose. Renature the proteins and block the membrane by incuba-
tion at 4 ° overnight in binding buffer [20 mM MOPS, pH 7.1, 100 mM

potassium acetate, 5 mM magnesium acetate, 5 mM DTT, 0.5% (w/v) BSA,
0.05% (v/v) Tween 20] containing 0.25% (v/v) Tween 20 and 5% (w/v)
milk. Alternatively, recombinant proteins to be tested can be spotted di-
rectly onto nitrocellulose. Spot small volumes of putative effectors (up to
2/~g) on small pieces of nitrocellulose and allow to dry at room temperature
for I hr. Block the membrane in binding buffer containing 5% (w/v) milk
at 4 ° for 1 hr in a small container.
To block nonspecific GTP binding, incubate the membrane in a small
volume (-<5 ml) of binding buffer containing 100 ~M GTP at 4 ° for 30
rain. Load recombinant GTPases with [~-32p]GTP or [~-32P]GDP, remove
unincorporated nucleotide, and quantitate complexed nucleotide as de-
scribed earlier. Add equal counts per minute (cpms) of loaded GTPases
to the blots at 4 ° and incubate for 10 min with rocking. Wash the blots
briefly (5-10 sec) with binding buffer until no further radioactivity is re-
moved. Analyze by fluorography with exposures of 1-2 hr and then over-
17 p. L. Bartel and S. Fields, Methods Enzymol.
254,
241 (1995).
i~ E. Manser, T. Leung, C. Monfries, M. Teo, C. Hall, and L. Lira,
J. Biol. Chem. 267,
16025 (1992).
12
PURIFICATION, MODIFICATION, AND REGULATION [l]
night. The [a-32p]GTP or [o~-32p]GDP will diffuse away from the proteins
over time, especially at room temperature, so the film exposures should be
done immediately after completion of the assay.
Coprecipitation Assay
Coprecipitation assays can be performed with a recombinant GST-
fusion protein, and glutathione-Sepharose beads, to study its interaction
with another recombinant protein or a protein expressed either ectopically

or endogenously in cells. 19 The interaction can be analyzed most easily by
SDS-PAGE and Coomassie staining if the proteins are of different sizes.
If there is an antibody against the protein to be precipitated or if a tagged
protein is precipitated, the interaction can be analyzed by immunoblotting;
an anti-GST antibody can be used to quantitate the amount of GST-fusion
protein bound to the beads. Either GST-TC10 bound to glutathione-
Sepharose can be used to assay its interaction with an effector protein
or a GST-fusion protein of the effector protein can be used to assay its
interaction TC10. Because there are currently no available antibodies
against TC10, either a tagged version of TC10 or [o~-32p]GTP-loaded TC10
needs to be used.
Procedure.
Exchange recombinant GST-TC10 into binding buffer (see
earlier discussion) with a PD10 or Centricep column. Bind 25-50 txg GST-
TC10 to 10/xl of glutathione-Sepharose beads (washed in binding buffer)
in a microcentrifuge tube at 4 ° for 1 hr. Wash excess GST-TC10 from
the beads once with binding buffer. Add an equimolar concentration of
recombinant, cleaved effector protein in a small volume (40-200 /zl) of
binding buffer and incubate at 4 ° for 1 hr. (Alternatively, GST-TC10 and
a cleaved effector protein can be added to the beads at the same time.)
Wash the beads three to five times with binding buffer. Add 10 /zl 2×
SDS-PAGE sample buffer to the beads, and fractionate the proteins by
SDS-PAGE. As controls, add effector to glutathione-Sepharose beads
and to glutathione-Sepharose beads bound to GST. For comparison, frac-
tionate the amount of GST-fusion coupled to the beads and effector added
to the beads.
To measure the interaction of TC10 with a GST-fusion of an effector
protein by scintillation counting, load recombinant, cleaved TC10 with
[o~-32P]GTP as described earlier. Incubate the loaded TC10 with glutathi-
one-Sepharose beads bound to a GST-fusion of the effector protein and

assay as described earlier. After washing the beads, cut the top of the
microcentrifuge tube and place the tube in a scintillation vial; fill the vial
~9 p. H. Warne, P. R. Viciana, and J. Downward,
Nature
364, 352 (1993).
[ 1 ] CHARACTERIZATION OF TC10 13
with scintillation fluid and quantitate. Alternatively, this interaction can be
assayed with recombinant or ectopically expressed, tagged TC10.
To affinity precipitate a protein from mammalian cells, lyse cells in 400
~1 of a cell lysis buffer with the cells on ice. Preclear the lysate with 0.5 ml
of glutathione-Sepharose beads (washed with lysis buffer) and 2.5 mg GST
at 4 ° for 1 hr. Add the cleared lysate to the glutathione-Sepharose beads
bound to GST-TC10 and assay as described previously; wash the beads
with lysis buffer.
Coimmunoprecipitation
To detect interaction of TC10 with a putative effector protein by co-
immunoprecipitation, 2° coexpress HA- or Myc-tagged TC10(Q75L) or
TC10(T31N) (as a negative control) with an effector protein fused to an-
other tag (HA or Myc) in NIH 3T3 or COS-7 cells. Two days after transfec-
tion, place the ceils on ice, wash once with PBS, and add 400/xl lysis buffer
[25 mM HEPES, pH 7.4, 300 mM NaC1, 1.5 mM MgC12, 0.5 mM DTT, 20
mM/Lglycerophosphate, 1 mM sodium vanadate, 1 mM PMSF, 20/xg/ml
aprotinin, 10/xg/ml leupeptin, and 0.1% (v/v) Triton X-100]. Scrape the
cells from the plate and centrifuge at 13,000g at 4 ° for 5 min. Remove 50 t~l
of each soluble lysate to determine protein expression by immunoblotting.
Incubate the remaining soluble supernatant with 3/xg 12CA5 or 4/xg 9El0
antibody at 4 ° for 1 hr. Add 30/xl protein A-Sepharose (washed with lysis
buffer) to 12CA5 immunoprecipitations or GammaBind Plus Sepharose to
9E10 immunoprecipitations and incubate at 4 ° for 1 hr. Wash the beads
three times with PBS containing 0.1% Triton X-100 and three times with

PBS. Add 30/xl 2× SDS-PAGE sample buffer to the beads. Fractionate
the expressed and immunoprecipitated proteins with 12% SDS-PAGE and
transfer to nitrocellulose for immunoblotting. To avoid detection of the
antibody used in the immunoprecipitation by the antimouse secondary
antibody, use 12CA5 or 9E10 coupled directly to horseradish peroxidase.
Competition Assays
To detect a low-affinity interaction between a GTPase and an effector
protein in vitro, a competition assay can be performed. 19 The assay is based
on the fact that epitopes on the GTPases to which effector proteins bind
overlap with the GAP-binding site. Thus, effector proteins inhibit GAP
activity. Because this inhibition is competitive, it can be used to assess the
affinity of the effector protein for the GTPase. The concentration of GAP
?~' E. Harlow and D. Lane, in "Using Antibodies: A Laboratory Manual," p. 223. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1999.
14 PURIFICATION, MODIFICATION, AND REGULATION
[1]
used in the assay is determined empirically so that in the absence of effector
protein, there is approximately 95% hydrolysis of GTP during the period
of the assay. The specificity of binding for an effector can be determined
by comparing this assay to various GTPases. The concentration of GAP
may have to be adjusted for each GTPase.
Procedure. To determine the lowest concentration of TC10 feasible for
this assay, load recombinant, cleaved TC10 with [T-32p]GTP as described
previously. Dilute the loaded TC10 to various concentrations. In a micro-
centrifuge tube, add 25 /xl 2× reaction buffer (50 mM MOPS, pH 7.1,
2 mM GDP, 10 mM MgC12, i mM sodium phosphate, 2 mM 2-mercaptoetha-
nol, and 0.1% (v/v) BSA), 20 /xl of the buffer in which the effector is
diluted, and 2.5/xl of diluted, loaded TC10 and incubate on ice for 20 min.
Add 2.5/xl of the buffer in which the GAP is diluted and incubate at 30 ° for
3 min. Filter bind 40/xl as described earlier and quantitate with scintillation

counting. Select a concentration of TC10 that yields approximately 3000-
5000 cpm for the competition assay; a concentration in the picomolar range
is ideal.
To determine the concentration of GAP to be used in the assay, add
25/xl 2× reaction buffer, 20/xl of the buffer in which the effector is diluted,
and 2.5/xl TC10 and incubate on ice for 20 min. Add 2.5/xl of diluted GAP
and incubate at 30 ° for 3 rain. Filter bind 40/xl as described earlier and
quantitate with scintillation counting. Plot GAP concentration vs the mean
cpm remaining complexed. Select a concentration of GAP that provides
95% hydrolysis of GTP.
The competition assay is performed as for the determination of GAP
concentration, using the concentrations of TC10 and GAP determined as
described previously. The effector is diluted to various concentrations and
incubated with TC10 on ice for 20 min prior to the addition of GAP. Plot
effector concentration vs the mean cpm remaining complexed. As controls,
the values obtained with TC10 alone, TC10 and GAP, and TC10 and
effector are compared to the results of the competition assay.
Acknowledgments
This work was supported by Grant CA40042 (to I.G.M.) and a University of Virginia
Pratt Fellowship (to C.L.N.).
[21 Rho/RhoGDI COMPLEX PURIFICATION 1 5
[2] Expression and Purification of
Rho / RhoGDI Complexes
By PAUL W. READ and ROBERT K. NAKAMOTO
Introduction
Recombinant protein production of Rho proteins and Rho guanine
dissociation inhibitor (RhoGDI) has resulted in a tremendous wealth of
information. To date, the majority of studies using recombinant Rho pro-
teins has utilized protein expressed in
Escherichia coli.

Relatively high yields
can be obtained, but the Rho protein is not posttranslationally modified.
Expression in eukaryotic cells provides posttranslational modifications of
the carboxyl-terminal CAAX sequence: transfer of a geranylgeranyl (or
farnesyl for some Rho proteins) group to the cysteine, proteolytic removal
of the final three residues, and carboxy methylation of the new terminal
cysteine. It appears that overexpression can overwhelm the processing
machinery, resulting in posttranslational modification on only a fraction of
the Rho protein. Prenylation appears to be the most important modification
and is required for full functionality, including membrane association and
binding to RhoGDI) 4
To study Rho/RhoGDl interactions, posttranslationally processed Rho
proteins have been purified from native sources 1'5-9 or eukaryotic expres-
sion systems such as Sf9 cells s'L°'~ and from
in vitro
translation of Rho
Y. Hori, A. Kikuchi, M. Isomura, M. Katayama, Y. Muira, H. Fujioka, K. Kaibuchi, and
Y. Takai, Oncogene 6, 515 (1991).
2 T. Mizuno, K. Kaibuchi, T. Yamamoto, M. Kawamura, T. Sakoda, H. Fujioka, Y. Matsuura,
and Y. Takai, Proc. Natl. Acad. Sci. U.S.A. 88, 6442 (1991).
3j. F. Hancock, K. Cadwallader, and C. J. Marshall, EMBO J. 10, 641 (1991).
4 j. F. Hancock, K. Cadwallader, H.
Paterson, and C. J. Marshall, EMBO J. 10, 4033 (1991).
Y. Fukumoto, K. Kaibuchi, Y. Hori, H. Fujioka, S. Araki, T. Ueda, A. Kikuchi, and Y.
Takai, Oncogene 5, 1321 (1990).
~' Y. Matsui, A. Kikuchi, S. Araki, Y. Hata, J. Kondo, Y.
Teranishi, and Y. Takai, MoL Cell
Biol. 10, 4116 (1990).
7 T. Ueda, A. Kikuchi, N. Ohga, J. Yamamoto, and Y. Takai, J. Biol. Chem. 265, 9373 (1990).
s M. J. Hart, Y. Maru, D. Leonard, O. N. Witte, T. Evans, and R. A. Cerione, Science 258,

812 (1992).
9 D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J.
Biol. Chem. 26'/, 22860 (1992).
~0 S. Ando, K. Kaibuchi, T. Sasaki, K. Hiraoka, T. Nishiyama, T. Mizuno, M. Asada, H. Nunoi,
I. Matsuda, Y. Matsuura, P. Polakis, F.
McCormick, and Y. Takai, J. Biol. Chem. 267,
25709 (1992).
~l T. K.
Nomanbhoy and R. A. Cerione, .1. Biol. Chem. 271, 10004 (1996).
Copyright © 20(~0 by Academic Press
All rights of reproduction in any form reserved.
METttODS IN ENZYMOLO(-;Y, VOL. 325 0076 6879/00 $30.00
16 PURIFICATION, MODIFICATION, AND REGULATION [2]
proteins in rabbit reticulocyte lysates supplemented with canine pancreatic
microsomal membranes. 3,a2 In addition, prokaryotic-derived Rho proteins
prenylated
in vitro
with recombinant geranylgeranyltransferase 13 or with
C-terminal truncation deletions with prenylated peptides added to mimic
full-length prenylated Rho proteins have also been used] 4
We have developed a coexpression system in
Saccharomyces cerevisiae
in which either a wild-type or constitutively active mutant Rho protein and
RhoGDI are coexpressed: one with a hexahistidine (His6) amino-terminal
tag and the other with a FLAG (DYKDDDK-) amino-terminal tag. 15 The
purification by sequential passage over a metal chelate column followed
by the anti-FLAG antibody column effectively prevents contamination by
endogenous yeast Rho or RhoGDI proteins. Because the Rho proteins are
isolated as a complex with RhoGDI, the Rho protein must be prenylated,
as RhoGDI association requires the geranylgeranyl moiety.l,3,1°,16 Expres-

sion of both proteins, particularly RhoGDI, is toxic to yeast and therefore
requires the use of a tightly regulated promoter such as
GALl.
Purification
of the complex from the yeast cytosol yields 100-300/zg of complex/liter
of culture, which is greater than 98% pure. The complexes have a 1:1
stoichiometry of Rho : RhoGDI without contamination from yeast homo-
logs and a 1 : 1 nucleotide : protein molar ratio. Unlike crude preparations
from tissue, the purified complex can be stoichiometrically ADP-ribosylated
by the
Clostridium botulinum
C3 exoenzyme and immunoprecipitated by
the 26-C4 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) made against the Rho insert helix (amino acids 124-136). 15,17 The
FLAG-RhoA/His6-RhoGDI complex has been used for structure determi-
nation by X-ray crystallography, signal transduction studies, and Rho pro-
tein activation studies. 1s'17 As expected, while bound to RhoGDI, RhoA
has kinetics of nucleotide exchange (3.3 × 10 4 sec 1 _+ 0.3 × 10 4 at 22 °)
and hydrolysis (0.45 × 10 -4 sec 1 _+ 0.02 x 10 4 at 22 °) significantly slower
than for free RhoA and consistent with the inhibitory properties of
RhoGDI. 15 Thus far, we have not observed any significant differences in
12 p. Lang, F. Gesbert, M. Delespine-Carmagnat, R. Stancou, M. Pouchelet, and J. Bertoglio,
EMBO J.
15, 510 (1996).
13 S. A. Armstrong, V. C. Hannah, J. L. Goldstein, and M. S. Brown,
J. Biol. Chem.
270,
7864 (1995).
14 A. R. Newcombe, R. W. Stockley, J. L. Hunter, and M. R. Webb,
Biochemistry

38, 6879
(1999).
is p. W. Read, X. Liu, K. Longenecker, C. G. DiPierro, L. A. Walker, A. V. Somlyo, A. P.
Somlyo, and R. K. Nakamoto,
Protein Sci.
9, 376 (2000).
16 j. F. Hancock and A. Hall,
EMBO J. 12,
1915 (1993).
17 K. Longenecker, P. Read, U. Derewenda, Z. Dauter, X. Liu, S. Garrard, L. Walker, A. V.
Somlyo, R. K. Nakamoto, A. P. Somlyo, and Z. S. Derewenda,
Acta Crystallogr.
D55,
1503 (1999).
[2] Rho/RhoGDl COMPLEX PURIFICATION 17
nucleotide binding or GTPase characteristics of the proteins if the amino-
terminal affinity tags are switched between the proteins or removed proteo-
lytically. In addition to RhoA, CDC42 and Racl and their constitutively
active mutants (G14V for RhoA and GI2V for CDC42 and Racl) have
been expressed successfully in this system.
Construction of Vectors
Construction of
YEpPG~,I/IpMA t
Expression Vector
The
GallO
promoter region ~s was inserted between the
EcoRI
and the
BarnHI

sites of the yeast 2-/~m shuttle vectors YEplac181
(LEU2)
and
YEplac195
(URA3). "~
In addition, the 3' half of the yeast
PMA1
gene,
including its transcription termination signal, was inserted between
BarnHI
and
XbaI. 2°
The
PMA1
segment included a
XhoI
site, which was created
by ligation of a linker in the
SalI
site 260 bases upstream of the
PMA1
termination codon. This created plasmid YEpPc;AUtpMA1 (Fig. 1A).
Insertion of Human cDNA into Yeast Expression Vectors
In order to optimize translational efficiency in yeast, a fragment was
cloned into the
BarnHI
site of YEpPc;Ac/tpMA1, which contained the se-
quence immediately upstream of the initiation codon and the first five
codons from the highly expressed yeast
PMA1

gene followed by His6 or
FLAG affinity tags (Fig. 1B). An
EagI
site following the tags provided the
cloning site for the Rho protein 2t or RhoGDI cDNA. When a protease
site was desired for removal of the affinity tags, the recognition sequence
for the rTEV protease (-DYDIPTTENLYFQG-) was added to the Rho
protein and RhoGDI cDNA, 3' of the
EagI
site, by PCR with extended
primers.
Expression and Purification of the RhoA/RhoGDI Complex
Yeast Growth
A leucine and uracil auxotrophic yeast strain is cotransformed with the
expression plasmids by standard methods. = We used strain SYI
(MATa,
is M. Johnston and R. W. Davis. MoL Cell. BioL 4, 1440 (1984).
~9 R. D. Gietz and A. Sugino, Gene 74, 527 (1988).
> R. K. Nakamoto, R. Rao, and C. W. Slayman, J. Biol. Chem. 266, 7940 (1991).
"~ H. F. Paterson, A. J. Self, M. D. Garrett, 1. Just, K. Aktories, and A. Hall, J. Cell Blot
111, 1001 (1990).
_,2 D. M. Becker and L. Guarente, Methods Enzyrnol. 194, 182 (1991).
18
PURIFICATION, MODIFICATION, AND REGULATION [2]
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[2] Rho/RhoGDl COMPLEX PURIFICATION 19
ura3-52, leu2-3,112, his4-619, sec6-4, GAL2°), but others can be used. Ini-
tially, an inoculum of a 50-ml culture of SC medium containing 2% glucose,
6.7 gm/liter yeast nitrogen base without amino acids (Difco Laboratories,
Detroit, MI), 30 mg/liter L-isoleucine, 150 mg/liter L-valine, 20 mg/liter
L-arginine, 20 mg/liter L-histidine hydrochloride, 30 mg/liter L-lysine hydro-
chloride, 20 mg/liter L-methionine, 50 mg/liter L-phenylalanine, 200 rag/
liter L-threonine, 20 mg/liter L-tryptophan, 30 mg/liter L-tyrosine, and 20
mg/liter adenine sulfate (uracil and leucine are omitted to provide auxotro-
phic selection of the plasmids23). The culture is grown for 24 hr at 25 ° with
vigorous shaking. This initial culture is used to inoculate 2.8-liter Fernbach
flasks containing 1 liter of the same minimal medium, except 2% glucose
is replaced with 2% raffinose. Raffinose is a poorly utilized carbon source
that neither induces nor represses the GALl promoter. To induce, galactose
is added and, unlike glucose, raffinose need not be removed. Growth is
allowed to continue until the optical density at the 650-nm wavelength
(OD650 ) reaches 1.0-1.2. If less than a milligram of purified complex is
required, protein expression is induced by the addition of 2% galactose in
the Fernbach flasks. If several milligrams are desired, 2 liters of culture is
used to inoculate a fermentor containing the same growth media [we use

a 19-liter capacity fermentor (Bellco, Vineland, N J) with a high-speed mixer
or large stir bar and aeration by an aquarium pump]. The doubling time
is approximately 4.5-5.0 hr. When the
0D650 nm
reaches 1.1-1.2, 2% galac-
tose is added, and the culture is allowed to grow for 7-8 hr or until growth
reaches late log, which usually occurs at an OD~s0 ,lm of 2.7 3.0.
Yeast are harvested by centrifugation at 1100g for 5 min at 4 °, washed
in 10 volumes of ice-cold 10 mM NAN3, and resuspended in 4 ml of 10 mM
NaN3 per gram of cells. Azide helps reduce proteolysis by stopping the
synthesis of ATP, which is required to activate cellular proteases. Cells are
flash frozen in liquid nitrogen and stored at -80 ° and can remain for several
months prior to protein purification. Routinely, 4-5 g wet cell weight/liter
of culture is obtained. We have observed that for strain SY1, the pellets
are tan colored when RhoGDI is expressed and white when not, whether
or not Rho protein is coexpressed.
Fractionation of Yeast
The cell walls are first digested for efficient lysis by treatment with
Zymolyase (ICN Biochemicals, Irvine, CA): 1.25 mg of Zymolase 20T
per gram wet cell weight of yeast and 50 /zM 2-mercaptoethanol final
concentration are added to 40 ml of spheroplasting buffer (2.8 M sorbitol,
23 F. Sherman, Methods Enzyrnol. 194, 3 (1991).
20 PURIFICATION, MODIFICATION, AND REGULATION [2]
100 mM KH2PO4, 10 mM NAN3, pH 7.4) and are incubated at 37 ° for
1 hr to activate the Zymolyase. The spheroplasting buffer (containing the
activated Zymolyase) is added in equal volume to the cell suspension and
is gently mixed at room temperature for 1 hr. The spheroplasts are sedi-
mented at 2800g for 10 min at 4 ° and resuspended in 4 ml of resuspension
buffer [50 mM Tris-base, 150 mM NaC1, 5 mM MgC12, 10% (v/v) glycerol,
pH 8.0 at 4°]. A protease cocktail is added consisting of 1 txg/ml leupeptin,

2 /xM pepstatin, 2 /xg/ml aprotinin, 40 /xg/ml benzamidine, and 1 mM
Pefabloc (or AEBSF; Pentapharm, Basil, Switzerland) (final concentra-
tions), and the suspension is subjected to two passes through a cooled
French press cell at 25,000 psi. Phenylmethylsulfonyl fluoride (1 mM freshly
dissolved in dimethyl sulfoxide) is added after the first pass. After a 5-min
centrifugation at 13,000g at 4 °, the supernatant is centrifuged at 240,000g
for 1 hr at 4 °. The Rho/RhoGDI complex is purified from the supernatant
(cytosolic fraction), and prenylated RhoA can be isolated from the mem-
brane fraction. Both cytosol and membrane fractions can be flash frozen
in liquid nitrogen and stored at -80 °. Cell lysis and fractionation require
5 hr.
Purification of the Rho/RhoGDl Complex
Purification of the complex via the amino-terminal tags involves sequen-
tial utilization of metal affinity resin (e.g., Ni-NTA resin from Qiagen,
Valencia, CA) and M2 anti-FLAG antibody (Sigma Chemicals, St. Louis,
MO) columns and requires 6 hr for a large-scale preparation. The column
material may be regenerated according to manufacturers' instructions and
reused several times without loss of effectiveness. The cytosolic fraction is
diluted i : 4 with Tris buffer (25 mM Tris-base, 150 mM NaC1, 5 mM MgCI2,
10% glycerol, pH 8.0) to decrease protein concentration to less than 2
mg/ml to reduce nonspecific protein interactions. The protein suspension
is incubated with 1 ml of equilibrated metal affinity resin per 250 mg of
cytosolic protein with gentle mixing for 45 min at room temperature. The
resin is sedimented by centrifugation at 700g for 2 minutes, packed into a
column, and washed with 10 bed volumes of Tris buffer. Bound protein is
eluted with metal elution buffer (50 mM Tris-base, 150 mM NaC1, 5 mM
MgCI2, 150 mM imidazole, pH 7.4) and collected in 2-ml fractions. The
absorbance at 280 nm of each fraction is measured. The peak fractions,
usually eluting in the second and third bed volume, are pooled and then
passed five times over an equilibrated M2 anti-FLAG antibody column

(0.7 ml bed volume per 250 mg of cytosolic protein). The column is then
washed with 10 bed volumes of M2 wash buffer (50 mM Tris-base, 150
mM NaC1, and 5 mM MgC12, pH 7.4) and eluted with four bed volumes