Preface GTPases are now recognized to regulate many different steps in mem- brane vesicular transport. They are involved in the assembly of vesicle coats (budding), movement along cytoskeletal elements, and in vesicle targeting and in fusion. They are clearly a key group of regulatory proteins that control transport through both the exocytic and endocytic pathways. GTPases involved in membrane transport include the Rab and ARF fami- lies, Sarl, and dynamin. Because these GTPases are switches, they function by either responding to or controlling the activity of a range of upstream and downstream effectors. These include posttranslational modifying enzymes (such as prenyltransferases and myristyltransferases), factors which effect guanine nucleotide binding ]guanine nucleotide dissociation inhibitors (GDIs) and guanine nucleotide exchange factors (GEFs)], and factors which stimulate guanine nucleotide hydrolysis [GTPase-activating proteins (GAPs)]. Moreover, they may also interact with motors and structural elements dictating vesicle and organelle function. The number of identified effectors directing or responding to transport GTPases is expanding rapidly. The purpose of this volume is to bring together the latest technologies that have developed over the past 5 years to study their function. Because each family contains a variety of isoforms, the techniques described for a particular GTPase family member are likely to be useful for other members of the same family. Moreover, the underlying conserved structural fold suggests that each of the various techniques are also applicable to other members of the larger superfamily of Ras-like GTPases. Given the abundance of both Rab and ARF GTPases and the intense interest of the cell biology community in their function, we have provided short editorial overviews for these two sections that describe the central features of their function and structural organization. We are extremely grateful to the many investigators who have gener- ously contributed their time and expertise to bring this wealth of technical experience into one volume. It should provide a valuable resource to ad- dress the many issues confronting our understanding of the role of these GTPases in cell biology. WILLIAM E. BALCH CHANNING J. DER ALAN HALL xvii Contributors to Volume 329 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. JOSEPH P. ALBANESI (51), Department of Pharmacology, University of Texas South- western Medical Center, Dallas, Texas 75390-9041 STEFAN ALBERT (6), Department of Molecular Genetics, Max Planck Institute for Biophys- ical Chemistry, GOttingen D-37070, Germany KmlLL ALEXANDROV (3), Department of Physical Biochemistry, Max Planck Insti- tute for Molecular Physiology, Dortmund 44202, Germany MEIR ARIDOR (45), Department of Cell Biol- ogy, The Scripps Research Institute, La Jolla, California 92037 LORRAINE M. ARON (23), Monoclonal Anti- body Facility, University of Georgia, Ath- ens, Georgia 30602 WILLIAM E. BALCH (1,2, 25, 45), Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CaliJbrnia 92037 MANUEL A. BARBIERI (16), Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 CHARLES BARLOWE (46), Department of Bio- chemistry, Dartmouth Medical School Hanover, New Hampshire 03755 BARBARA BARYLKO (51), Department of Pharmacology, University of Texas South- western Medical Center, Dallas, Texas 75390-9041 CRESTINA L. BEITES (52), Programme in Cell Biology, Hospital for Sick Children, De- partment of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G lX8 WILLIAM J. BELDEN (46), Department of Bio- chemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 SOPHIE BI~RAUD-DUFOUR (25, 28), Depart- ment of Molecular and Cell Biology, The Scripps Research Institute, La Jolla, Califor- nia 92037 KUN BI (38), Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235 DERK D. BINNS (51), Department of Pharma- cology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041 JAMES E. CASANOVA (23, 27), Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 DAN CASSEL (33, 34), Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel PHILIPPE CHAVRIER (29), Institut Curie- Section Recherche, CNRS UMR 144, Paris' Cedex 05, France WEI CHEN (18, 19), National Center for Ge- nome Resources, Santa re, New Mexico 87505 SAVVAS CHRISTOFORIDIS (14), Laboratory of Biological Chemistry, Medical School University of loannina, loannina 45110, Greece SHAMSHAD COCKCROFT (38), Department of Physiology, University College, London WC1E6JJ, United Kingdom EDNA CUKIERMAN (33), Department of Biol- ogy, Technion-lsrael Institute of Technol- ogy, Haifa 32000, Israel MICHAEL P. CZECH (30), Program in Molecu- lar Medicine and Department of Biochemis- try and Molecular Biology, University of xi xii CONTRIBUTORS TO VOLUME 329 Massachusetts Medical School, Worcester, Massachusetts' 01605 HANNA DAMKE (47), Department of Cell Biol- ogy, The Scripps Research Institute, La Jolla, California 92037 PmTRO DE CAMILLI (50), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510 MARIA ANTONIETTA DE MATFEIS (42), De- partment of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria lmbaro, Chieti 66030, Italy MAGDA DENEKA (13), Department of Cell Bi- ology, Utrecht University School of Medi- cine, Utrecht 3584 CX, The Netherlands JULIE G. DONALDSON (26), Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0301 MATTHEW T. DRAKE (40), Department of In- ternal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 ROCKFORD K. DRAPER (39), Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083-0688 LI-L1N Du (11), Department of Molecular Biophysics and Biochemistry, Yale Univer- sity School of Medicine, New Haven, Con- necticut 06520-8002 STEVEN DUNKELBARGER (12), Department of Biochemistry and Molecular Biology, Uni- formed Services University of the Health Sciences, Bethesda, Maryland 20814 ARNAUD ECHARD (17), Laboratoire M(ca- nismes MolOculaires du Transport Intracel- lulaire, UMR CNRS 144, lnstitut Curie, Paris Cedex 05, France AHMED EL MARJOU (17), Service des Pro- tdines Recombinantes, UMR CNRS 144, In- stitut Curie, Paris Cedex 05, France YAN FENC (19), Department of Chemistry and Cell Biology, Harvard Medical School, Bos- ton, Massachusetts 02115 SUSAN FERRO-Nov1cK (24), Department of Cell Biology, Boyer Center for Molecular Medicine, Howard Hughes Medical Insti- tute, Yale University School of Medicine, New Haven, Connecticut 06510 MICHEL FRANCO (29), Institut de Pharmacolo- gie, Mol~culaire et Cellulaire, CNRS UPR 411, Valbonne 06650, France SCOTT R. FRANK (27), DNAX, Palo Alto, Cal- ifornia 94304 JOHANNA FURUHJELM (20), Institute of Bio- technology, PB56, University of Helsinki, Helsinki FIN 00014, Finland THIERRY GALLI (21), Trafic Membranaire et Plasticitd Neuronale, INSERM U536, Insti- tut Curie, Paris Cedex 05, France DIETER GALLWITZ (6), Department of Molec- ular Genetics, Max Planck Institute for Bio- physical Chemistry, GOttingen D-37070, Germany JAMES R. GOLDENRING (23), Institute for Mo- lecular Medicine and Genetics, Depart- ments of Medicine, Surgery, Cellular Biol- ogy and Anatomy, Medical College of Georgia and Augusta Veterans Affairs Med- ical Center, Augusta, Georgia 30912-3175 ROGER S. GOODY (3), Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund 44202, Germany BRUNO GOUD (17), Laboratoire MOcanismes Moldculaires du Transport lntracellulaire, UMR CNRS 144, lnstitut Curie, Paris Cedex 05, France A. GUMUSBOGA (16), Department of Cell Bi- ology and Physiology, Washington Univer- sity School of Medicine, St. Louis, Mis- souri 63110 WEI Guo (12), Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002 HISANOR1 HORIUCHI (15), Department of Ge- riatric Medicine, Kyoto University Hospital, Kyoto City 606-01, Japan TONOHUAN HU (39), Department of Molecu- lar Biology, University of Texas Southwest- ern Medical Center, Dallas, Texas 75390- 9148 CONTRIBUTORS TO VOLUME 329 xiii CHUN-FANG HUANG (43), Institute of Molecu- lar Medicine College of Medicine, National Taiwan University, Taipei, Taiwan 100, Re- public of China IRIT HUBER (33, 34), Department of Biology, Technion-lsrael Institute of Technology, Haifa 32000, Israel ROBERT TOD HUDSON (39), Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medi- cine, Cleveland, Ohio 44106-4900 WALTER HUNZIKER (22), Institute for Molecu- lar and Cell Biology, Singapore 117609, Re- public of Singapore CATHERINE L. JACKSON (31), Service de Bio- chimie et Gdn~tique Mol~culaire, CEA/ Saclay, Gif-sur-Yvette, Cedex F-91191, France TREVOR R. JACKSON (37), Department of He- matology, Royal Free and University Col- lege Medical School Royal Free Campus, London NW3 2AF, United Kingdom GERALD C. JOHNSTON (34), Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 MANDY JONGENEELEN (13), Department of Cell Biology, Utrecht University School of Medicine, Utrecht 3584 CX, The Nether- lands" JES K. KLARLUND (30), Ophthalmology and Visual Sciences Research Center, University of Pittsburgh School of Medicine, Pitts- burgh, Pennsylvania 15213 STUART KORNFELD (40), Department oflnter- nal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 NICHOLAS T. KTISTAKIS (38), Department of Signaling, Babraham Institute, Cambridge CB2 4AG, United Kingdom LYNNE A. LAP1ERRE (23), Institute for Molec- ular Medicine and Genetics, Departments of Medicine, Surgery, Cellular Biology and Anatomy, Medical College of Georgia and Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912-3175 ANTHONY LEE (48), Department of Bio- chemistry and Biophysics, University of Pennsylvania School of Medicine, The Johnson Research Foundation, Philadel- phia, Pennsylvania 19104-6059 FANG-JEN S. LEE (43), Institute of Molecular Medicine College of Medicine, National Taiwan University, Taipei, Taiwan 100, Re- public of China MARK A. LEMMON (48), Department of Bio- chemistry and Biophysics, University of Pennsylvania School of Medicine, The Johnson Research Foundation, Philadel- phia, Pennsylvania 19104-6059 ROCER LIPP~ (15), Max Planck Institute for Molecular Cell Biology and Genetics, Euro- pean Molecular Biology Laboratory, Hei- delberg 69117, Germany DANIEL LOUVARD (21), Morphogen~se et Sig- nalisation Cellulaires, URM 144, lnstitut Curie, Paris Cedex 05, France VARDIT MAKLER (33), Department of Biol- ogy, Technion-lsrael Institute of Technol- ogy, Haifa 32000, Israel WILLIAM A. MALTESE (4), Department of Biochemistry and Molecular Biology, Med- ical College of Ohio, Toledo, Ohio 43614- 5804 ANNE-MARm MARZESCO (21), Morphogenkse et Signalisation Cellulaires, URM 144, Insti- tut Curie, Paris Cedex 05, France JEANNE MATTESON (2), Departments of Cell and Molecular Biology, The Scripps Re- search Institute, La Jollu, California 92037 Kolcnl MIURA (37), Laboratory of Cellular Oncology, Division of Basic Sciences, Na- tional Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 KARIN MOHRMANN (13), Department of Cell Biology, Utrecht University School of Medi- cine, Utrecht 3584 CX, The Netherlands JON S. MORROW (42), Departments of Pathol- ogy, and Molecular, Cellular, and Develop- mental Biology, Yale University, New Ha- ven, Connecticut 06510 xiv CONTRIBUTORS TO VOLUME 329 JOEL MOSS (32, 35, 44), Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Insti- tutes of Health, Bethesda, Maryland 20892 BRYAN D. MOYER (1, 2), Departments of Cell and Molecular Biology, The Scripps Re- search Institute, La Jolla, California 92037 AMY B. MUHLBERG (47), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 FUM1KO NAGANO (8), Department of Molecu- lar Biology and Biochemistry, Osaka Uni- versity Graduate School of Medicine/ Faculty of Medicine, Osaka 565-0871, Japan HIROYUKI NAKANISHI (7), Department of Mo- lecular Biology and Biochemistry, Osaka University Graduate School of Medicine/ Faculty of Medicine, Osaka 565-0871, Japan JENNIFER NAVARRE (23), Institute for Molecu- lar Medicine and Genetics, Departments of Medicine, Surgery, Cellular Biology and Anatomy, Medical College of Georgia and Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912-3175 WALTER NICKEL (41), Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universitdt, Heidelberg D-69120, Germany PETER NOVICK (11, 12), Department of Cell Biology, Yale University School of Medi- cine, New Haven, Connecticut 06520- 80O2 SATOSHI ORITA (10), Discovery Research Laboratories, Shionogi and Company, Limited, Osaka 565-0871, Japan JEAN H. OVERMEYER (4), Department of Bio- chemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804 GUSTAVO PACHECO-RODRIGUEZ (32, 44), Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 X1Ao-RONG PENG (52), Programme in Cell Biology, Hospital for Sick Children, De- partment of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1X8 JOHAN PERANEN (20), Institute of Biotechnol- ogy, PB56, University of Helsinki, Helsinki FIN 00014, Finland PETER J. PETERS (22), Dutch Cancer Research Institute, Amsterdam, The Netherlands AYNE PEYROCHE (31), Service de Biochimie et GOn~tique Mol~culaire, CEA/Saclay, Gif-sur- Yvette, Cedex F-91191, France ELAH PICK (33), Department of Biology, Technion-lsrael Institute of Technology, Haifa 32000, Israel PAK PHI POON (34), Departments of Microbi- ology and Immunology, Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 RICHARD T. PREMONT (36), Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 BARRY PRESS (19), Dana Farber Cancer Insti- tute, Harvard Medical School, Boston, Mas- sachusetts 02115 HARISH RADHAKRISHNA (26), Department of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0363 PAUL A. RANDAZZO (37), Laboratory of Cellular Oncology, Division of Basic Sci- ences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 RICHARD L. ROBERTS (16), Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 SYLVIANE ROBINEAU (28), Institut de Pharma- cologie Mol~culaire et Cellulaire, CNRS, Valbonne 06560, France MICHAEL G. ROTH (38), Department of Bio- chemistry, University of Texas Southwest- ern Medical Center, Dallas, Texas 75235 LILAH ROTHEM (33), Department of Biology, Technion-lsrael Institute of Technology, Haifa 32000, Israel CONTRIBUTORS TO VOLUME 329 xv MIRIAM ROTMAN (33), Department of Biol- ogy, Technion-lsrael Institute of Technol- ogy, Haifa 32000, Israel ANJA RUNGE (15), Max Planck Institute for Molecular Cell Biology and Genetics, Euro- pean Molecular Biology Laboratory, Hei- delberg 69117, Germany MICHAEL SACHER (24), Department of Cell Biology, Boyer Center for Molecular Medi- cine, Yale University School of Medicine, New Haven, Connecticut 06510 LORRAINE C. SANTY (27), Department of Cell Biology, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908 TAKUYA SASAKI (8, 9, 10), Department of Bio- chemistry, Tokushima University School of Medicine, Tokushima 770-8503, Japan AXEL J. SCHEIDIG (3), Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology', Dortmund 44202, Germany SANDRA L. SCHMID (47), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 SANJA SEVER (47), Department of Cell Biol- ogy, The Scripps Research Institute, La Jolla, California 92037 HIROMICH1 SHIRATAK1 (9), Division of Molec- ular and Cell Biology, Institute for Medical Science, Dokkyo University School of Med- icine, Mibu 321-0293, Japan ASSIA SHISHEVA (5), Department of Physiol- ogy, Wayne State University School of Med- icine, Detroit, Michigan 48201 STEVEN SHOLLY (47), Department of Cell Bi- ology, The Scripps Research Institute, La Jolla, California 92037 DIXIE-LEE SHURLAND (49), Department of Bi- ological Chemistry, University of Califor- nia, School of Medicine, Los Angeles, Cali- fornia 90095-1737 RICHARD A. SINGER (34), Department of Bio- chemistry and Molecular Biology, Dalhou- sie University, Halifax, Nova Scotia, Can- ada B3H 4H7 VLADIMIR I. SLEPNEV (50), Department of Cell Biology, Yale University School of Medi- cine, New Haven, Connecticut 06510 ELENA SMIRNOVA (49), Department of Bio- logical Chemistry, University of California, School of Medicine, Los Angeles, Califor- nia 90095-1737 PHILIP D. STAHL (16), Department of Cell Bi- ology and Physiology, Washington Univer- sity School of Medicine, St. Louis, Mis- souri 63110 YOSHIMI TAKAI (7, 8, 9, 10), Department of Molecular Biology and Biochemistry, Osaka University Medical School, Osaka 700-8558, Japan KOHJl TAKEI (50), Department of Biochemis- try, Okayama University School of Medi- cine, Okayama-shi, Okayama 700-8558, Japan DANIEL R. TERBUSH ([2), Department of Bio- chemistry and Molecular Biology, Uni- formed Services University of the Health Sciences, Bethesda, Maryland 20814 WILLIAM S. TRIMBLE (52), Programme in Cell Biology, Hospital for Sick Children, De- partment of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G lX8 ALEXANDER M. VAN DER BLIEK (49), Depart- ment of Biological Chemistry, University of California, School of Medicine, Los Angeles, California 90095-1737 PETER VAN DER SLUIJS (13), Department of Cell Biology, Utrecht University School of Medicine, Utrecht 3584 CX, The Nether- lands" MARTHA VAUGHAN (32, 35, 44), Pulmonary- Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 NICOLAS VITALE (35, 36), Center de Neuro- chimie, 1NSERM U-338, Strasbourg, Cedex 67084, France ANGELA WANDINGER-NEss (18, 19), Depart- ment of Pathology, University of New Mex- ico Health Sciences Center, Albuquerque, New Mexico 87131 xvi CONTRIBUTORS TO VOLUME 329 DALE E. WARNOCK (47), Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 JACOUES T. WEISSMAN (45), Department of Cell Biology, The Scripps Research Insti- tute, La Jolla, California 92037 FELIX T. WIELAND (41), Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universiti~t, Heidelberg D-69120, Germany ELKE WILL (6), Department of Molecular Genetics, Max Planck Institute for Bio- physical Chemistry, GOttingen D-37070, Germany AHMED ZAHRAOUI (21), Morphogendse et Signalisation Cellulaires, URM 144, Institut Curie, Paris Cedex 05, France MAR1NO ZERIAL (14, 15), Max Planck Insti- tute for Molecular Cell Biology and Genet- ics, European Molecular Biology Labora- tory, Heidelberg 69117, Germany YUNXIANG ZHU (40), Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 JAY ZIMMERMAN (19), University of Chicago, Pritzker School of Medicine, Chicago, Illi- nois 60610 [1] Rab STRUCTURE-FUNCTION OVERVIEW 3 [1] Structural Basis for Rab Function: An Overview By BRYAN D. MOYER and WILLIAM E. BALCH Rab proteins, members of the Ras superfamily of low molecular weight GTP-binding proteins (-20-25 kDa), modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways, a 3 Simi- lar to Ras, Rab GTPases cycle between active, GTP-bound and inactive, GDP-bound states. 4 This brief introductory chapter summarizes Rab struc- ture-function relationships in the context of membrane trafficking and serves as a prelude for the accompanying chapters, which describe specific methods for elucidating Rab function. The unifying theme in research elucidating Rab structure-function rela- tionships has been the Rab GTPase cycle model (Fig. 1) (reviewed in Refs. 1, 2, and 4). In the cytosol, Rab proteins are maintained in the GDP-bound state by interaction with a GDP dissociation inhibitor (GDI). 5 GDI delivers Rab-GDP to donor membranes where GDI may be displaced by a GDI displacement factor (GDF). 6 Subsequently, a guanine nucleotide exchange factor (GEF) is believed to stimulate exchange of GDP for GTP. 7,8 Trans- port intermediates containing activated Rab bud from donor membranes, where Rab-GTP recruits effector molecules required for trafficking to acceptor compartments. 1,3 Recent studies suggest that Rab effectors regu- late the motility of transport intermediates along cytoskeletal elements and mediate the docking/fusion of transport intermediates with acceptor membranes. 9-~2 Prior to or concomitant with membrane docking and fusion, a GTPase activating protein (GAP) is thought to stimulate Rab-mediated hydrolysis of GTP to GDP and recruited effector molecules dissociate from 1 j. S. Rodman and A. Wandinger-Ness, J. Cell Sci. 113, 183 (2000). 20. Martinez and B. Goud, Biochim. Biophys. Acta 14t)4, 101 (1998). 3 F. Schimm611er, I. Simon, and S. R. Pfeffer, J. Biol. Chem. 273(35), 22161 (1998). 4 g. M. Olkkonen and H. Stenmark, Int. Rev. Cytol. 176, 1 (1997). 5 S K. Wu, K. Zeng, I. A. Wilson, and W. E. Balch, Trends Biochem. Sci. 21, 472 (1996). A. B. Dirac-Svejstrup, T. Sumizawa, and S. R. Pfeffer, EMBO J. 16(3), 465 (1997). T. Soldati, A. D. Shapiro, A. B. D. Svejstrup, and S. R. Pfeffer, Nature 369, 76 (1994). O. Ullrich, H. Horiuchi, C. Bucci, and M. Zerial, Nature 368, 157 (1994). A. Echard, F. Jollivet, O. Martinez, J J. Lacap6re, A. Rousselet, I. Janoueix-Lerosey, and B. Goud, Science 279, 580 (1998). 10 S. Christoforids, H. M. McBride, R. D. Burgoyne, and M. Zerial, Nature 397, 621 (1999). l~ S. R. Pfeffer, Nat. Cell Biol. 1, El7 (1999). 1~- M. G. Waters and S. R. Pfeffer, Curr. Opin. Cell Biol. 11, 453 (1999). Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. METHODS 1N ENZYMOLOGY, VOL. 329 0076-6879/00 $35.(1(/ 4 Rab GTPases [ 1 ] Donor ~ Acceptor Membrane Membrane >// °°P 3TP Pi ~D Flo. 1. Model of the Rab GTPase cycle. Rab-GDP/GDI complexes are targeted to donor membranes where GDF displaces GDI and a GEF stimulates Rab-GDP/GTP exchange (step 1). Transport intermediates containing Rab-GTP bud from donor membranes. Rab- GTP recruits effector molecules, which mediate the migration, docking, and fusion of transport intermediates to acceptor membranes (step 2). GTP is hydrolyzed to GDP by a GAP and effector molecules dissociate from Rab (step 3). GDI is recruited by RRF and extracts Rab-GDP from acceptor membranes for initiation of another round of the Rab GTPase cycle (step 4). See text for complete details. GDI, GDP dissociation inhibitor; GDF, GDI displacement factor; GEF, guanine nucleotide exchange factor; GAP, GTPase activating protein; RRF, Rab recycling factor. Rab. 13 GDI, recruited to membranes by a putative Rab recycling factor (RRF), 14 then extracts Rab-GDP from acceptor membranes and the Rab-GDP/GDI complex recycles to donor membranes for initiation of another round of transport. Rab GTPases contain conserved and unique sequence elements that mediate function, including GDP/GTP binding, subcellular targeting, and 13 V. Rybin, O. Ullrich, M. Rubino, K. Alexandrov, I. Simon, M. C. Seabra, R. Goody, and M. Zerial, Nature 383, 266 (1996). ~4 p. Luan, W. E. Balch, S. D. Emr, and C. G. Burd, J. Biol. Chem. 274(21), 14806 (1999). [ 1 ] Rab STRUCTURE-FUNCTION OVERVIEW 5 PM1 G1 ~t3 switch II switch I Rab CDR FIG. 2. Crystal structure of Rab3A. Pertinent structure elements that mediate Rab function are labeled and discussed in the text. N, N terminus; C, C terminus (site of geranylgeranyl lipid addition); Rab CDR, Rab complimentarity-determining region (site for specific effector binding); G1-G3, guanine base-binding motifs; PM1-3: phosphate/magnesium-binding motifs; GTP, guanosine triphosphate; Mg 2+, divalent magnesium ion; switch f-II: regions undergoing large conformational changes during GTP binding and hydrolysis. effector recognition (Fig. 2). 4 Rab proteins contain three highly conserved guanine base-binding motifs (termed G1 to G3) which mediate guanine nucleotide binding and three highly conserved phosphate/magnesium-bind- ing motifs (termed PM1 to PM3), which bind and coordinate a divalent magnesium ion with the/3- and y-phosphates of GTP. On GAP-stimulated hydrolysis of GTP to GDP and loss of the terminal phosphate group, two regions in spatial proximity to the y-phosphate, termed switch I (also called the effector domain) and switch II, undergo dramatic conformational changes, which result in reduced affinity for bound effector molecules and Rab inactivation. During Rab reactivation, GEF-stimulated conformational changes in the switch I, switch II, and P loop regions facilitate extrusion of GDP and incorporation of GTP. The C-terminal regions of Rab proteins are highly divergent and contain two structural elements dictating function. First, the extreme C termini [...]... the conserved switch I and switch II regions and second in nonconserved regions at the N terminus, central region, and C terminus These later hypervariable regions coalesce into a deep pocket termed a Rab complementarity-determining region (Rab CDR) Because Rab CDRs are not conserved between family members, they are proposed to determine the specific interaction between individual GTP-bound Rab proteins... occurring when nucleotides associate with or dissociate from the active site However, there may also be changes in the nucleotide fluorescence when other proteins interact with the GTPases, as first seen in the case of Ran and its exchange factor RCC17 and subsequently on interaction of complexes between Ras and mant nucleotides with GTPase activating proteins (GAPs) 9,1° More recently, the fluorescence mant-nucleotide... proteins and their effectors 15We are currently at a pivotal point in our understanding of the Rab GTPase family, which is now comprised of more than 40 members 1'4 Genetic and biochemical methodologies are rapidly revealing the identity of novel Rab effector molecules However the function of these effector molecules, in many instances, remains to be determined 15 C Ostermeier and A T Brunger, Cell 96,... complementarity-determining region (Rab CDR) Because Rab CDRs are not conserved between family members, they are proposed to determine the specific interaction between individual GTP-bound Rab proteins and their effectors 15We are currently at a pivotal point in our understanding of the Rab GTPase family, which is now comprised of more than 40 members 1'4 Genetic and biochemical methodologies are rapidly... doubly dansylated Rab7 protein as confirmed by mass spectrometry and fluorescence yield measurements Excitation and emission scans of the labeled protein revealed that fluorescence could be excited either directly at 333 nm or via fluorescence resonance energy transfer from tryptophan excited at 295 nm This method has general applicability and has also been successfully used for labeling of Rab5, Ypt7,... thoroughly vortex the LipofectAMINE transfection reagent before use to resuspend lipids that settle during storage Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 50-60% and get 5- to 20-fold overexpression of protein compared to the endogenous pool, as determined by immunoblotting Transfection efficiencies decrease with increasing plasmid size and DNA/ lipid... GDP Labeled protein is stored in multiple aliquots at - 8 0 ° The efficiency of labeling is determined by mass spectrometry and fluorescent yield measurements Results Preparation ofDansyl-Labeled Rab7 The C terminus of Rab GTPases is known not to have a definite structure in solution However, logic demands that the two C-terminal cysteines must be precisely positioned for the prenyl transfer reaction... protein components in the regulation of vesicular transport, ~ and as in other systems, signals must first be found before such interactions can be investigated In this chapter, we describe several examples of fluorescence signals that can be used for such studies As a model system we have chosen Rab7, a small G T P a s e involved in the biogenesis of late endosomes and lysosomes 2'3 We have investigated... complex, since the fluorescence increase on interaction with REP-1 was much smaller than in the case of R a b 7 - m a n t G D P However, as described below, this signal was adequate for transient kinetic experiments The interaction of Rab7 and REP-1 can also be monitored using the fluorescence of two dansyl labels covalently attached at the C terminus of Rab7 (method described below) There is a large... the peptide bond 14 The only requirement for the ligation reaction is the presence of the N-terminal cysteine to the target peptide Methods Vector Construction, Protein Expression, Purification, and Ligation We first generate an expression vector for C-terminal fusion of Rab7AC6 with intein by PCR amplifying the coding sequence of the former The 3' oligonucleotide is designed in such a way that the . pocket termed a Rab complementarity-determining region (Rab CDR). Because Rab CDRs are not conserved between family members, they are proposed to determine the specific interaction between individual. pocket termed a Rab complementarity-determining region (Rab CDR). Because Rab CDRs are not conserved between family members, they are proposed to determine the specific interaction between individual. methodologies are rapidly revealing the identity of novel Rab effector molecules. However the function of these effector molecules, in many instances, remains to be determined. 15 C. Ostermeier