MEMBRANES IN GROWTH AND DEVELOPMENT

Proceedings of the International Conference on Biological Membranes

June 15-19,1981 Crans-sur-Sierre, Switzerland

Editors

Joseph F. Hoffman Gerhard H. Giebisch Department of Physiology

Yale University New Häven, Connecticut

Liana Bolis Department of General Physiology

University of Messina Sicily, Italy

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Library of Congress Cataloging in Publication Data

Main entry under title:

Membranes in growth and development.

(Progress in clinical and biological research ; v. 91)

Papers presented at the Biennial International Conference on Biological Membranes.

Includes index and bibliographies. 1. Cell membranes —Congresses. 2. Developmental

cytology —Congresses. 3. Growth —Congresses. 4. Cellular control mechanisms-Congresses. I. Hoffman, Joseph F. II. Giebisch, Gerhard H. III. Bolis, Liana. IV. International Conference on Biological Membranes (1981 : Crans, Switzerland) V. Series. [DNLM:1. Cell membranes —Physiology — Congresses. W1 PR668E v. 91 / QH 601 C747m 1981] QH601.M4823 574.87'5 82-7178 ISBN 0-8451-0091-2 AACR2

Acknowledgments We wish to express our thanks for the support given by the following:

Abbott Laboratories (USA) Biogen (Switzerland) Burroughs Wellcome Co. (USA) Community of Chermignon (Switzerland) Du Pont (Switzerland) Fidia Research Laboratories (Italy) Golf Club de Crans-sur-Sierre (Switzerland) Hoechst Roussel Pharmaceuticals (USA) ICI Americas, Inc. (USA) Interpharma (Switzerland) Lirca Laboratories (Italy) Merck, Sharp & Dohme (USA) Miles Laboratories, Inc. (USA) Paul-Martin Stiftung (GR) Synthelabo (France) Tracor Analytic (USA)

Contents

Contributors xi Preface

J.F. Hoffman, G.H. Giebisch, and L Bolis xix

SECTION I. BIOSYNTHESIS OF MEMBRANE PROTEINS

The Signal Hypothesis in Bacteria Scott D. EmrandThomas J.Silhavy 3 Studies off Export off Protein in Escherichia coli Linda L Randall 15 Assembly of Membrane Proteins David I. Meyer, Sune Kvist, and Bernhard Dobberstein 23 Biogenesis of Mitochondrial Membrane Proteins Martin Teintze, Bernd Hennig, Manfred Schleyer, Bernd Schmidt, and Walter Neupert 37 Import of Cytoplasmically Synthesized Proteins Into Yeast Mitochondria Graeme A. Reid and Gottfried Schatz 49 Synthesis of the Erythrocyte Anion Transport Protein in Bone Marrow Erythroid Cells B. M.J. Foxwell and M.J.A. Tanner 59 Regulation by Turnover of Na,K-ATPase in HeLa Cells LR. Pollack, E.H. Täte, and J.S. Cook 71 Surface and Cytoplasmic Domains in Polarized Epithelial Cells Hubert Reggio, Evelyne Coudrier, and Daniel Louvard 89 Isolation and Properties of Spectrin Protomers V. Rudioff, F. Herbst, R. Passing, and D. Schubert 107 Erythrocyte Membrane Protein Deficiency in a Human Hemolytic Anemia: Viscometric- Diffractometric Evaluation (Ektacytometry) C. J.Feo 121

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viii / Contents

SECTION II. MEMBRANE PROCESSES IN FERTILIZATION AND CELL DIVISION

The Role of Calcium in Meiosis Reinitiation P. Guerrier, M. Moreau, L Meijer, G. Mazzei, J.P. Vilain, and F. Dube 139 Interaction of Steroids and Growth Factors With the Plasma Membrane in the Induction of Oocyte Maturation in Xenopus Laevis James L Maller 157 Intracellular Calcium and Fertilization: Role of the Cation and Regulation of Intracellular Calcium Levels David Epel, George Perry, and Tobias Schmidt 171 Some Aspects of Fertilization in Marine Invertebrates Christian Sardet, Daniele Carre, Marie Paule Cosson, Jacky Cosson, Patrick Chang, Patrick Payan, and Jean Pierre Girard 185 Cation Transport and Growth Control in Neuroblastoma Cells in Culture S.W. de Laat, J. Boonstra, W H . Moolenaar, C.L Mummery, P.T. van der Saag, and E.J.J. van Zoelen 211

SECTION III. DEVELOPMENT, REGENERATION, AND METABOLISM OF NERVOUS TISSUE

Immunological Identification of an Intracellular Postsynaptic Protein at the Frog Neuromuscular Synapse Steven J. Bürden 239 The Reorganization of Neuromuscular Junctions Düring Development in Rats Richard A.D. O'Brien, Anna J.C. Östberg, and Gerta Vrbovä. 247 Synthesis, Transport, and Fate of Acetylcholinesterase and Acetylcholine Receptors in Cultured Muscle Richard L Rotundo and Douglas M. Fambrough 259 The Acetylcholine Receptor and Its Membrane Localization Robert E. Oswald 287 Synapse Reformation and Repression in Muscle Reinnervation: An Evaluation of Endogenous and Exogenous Influences on Nerve Regeneration A. Gorio, M.G. Nunzi, P. Polato, and R. Zanoni 299 Gangliosides and Neuronal Cell Differentiation Alberta Leon, Gino Toffano, Alfredo Gorio, and Giovanna Ferrari 311 Pharmacological Block of Nerve Conduction Affects Electrical Properties of Muscle Membranes D. Conte Camerino, S.H. Bryant, and D. Mitolo-Chieppa 321

Contents / ix

A Polypeptide Involved in Membrane Transport of Substrate for Glucose-6-Phosphohydrolase Michael A.Zoccoli and Manfred L Karnovsky 329 Neuronal Interactions Düring Mammalian Brain Development: An In Vitro Study Umberto di Porzio and Mariela Estenoz 337 Brain and Nutrition: The Role of Taurine Gerald E. Gaull, John A. Sturman, Guang Y. Wen, and Henryk M. Wisniewski 349

SECTION IV. ROLE OF THE PLASMALEMMAL AND EXTRACELLULAR MATRIX IN TISSUE ORGANIZATION

Molecular Steps in the Action and Regulation of Epidermal Growth Factor: A Cellular Mitogen J. Schiessinger, A.B. Schreiber, I. Lax, Y. Yashuv-Gan, TA. Libermann, G.M. Hillman, A. Levi, and Y. Yarden 359 Control of Cell Growth and Development by the Antagonistic Effects of Cell-Cell Contact and Serum Components K.L. Caldwell and L. Glaser 373 Importance of Cell Shape for the Hormone Responsiveness of Rabbit Mammary Cells in Primary Cultures J.-P. Kraehenbühl, Y. Suard, L Racine, and M.T Häuptle 389 Thyroid Cell Polarization in Culture and the Expression of Specialized Functions M. Chambard, J. Gabrion, B. Verrier, and J. Mauchamp 403 Plasmalemmal Glycoproteins and Basal Lamina: Involvement in Pancreatic Morphogenesis J.D.Jamieson 413 The Extracellular Matrix and Kidney Differentiation Peter Ekblom, Lauri Saxen, and Rupert Timpl 429

V. MEMBRANE TÜRNOVER AND ENDOCYTOSIS

Studies on the Biosynthesis and Structure of Sindbis Virus High Mannose Oligosaccharides Paul H.Atkinson and John Hakimi 445 Metabolism of Plasma Membrane Glycoconjugates in Hepatic Cells of the Rat and After Transfer to Mouse Fibroblasts Heinz Baumann and Darrell Doyle 459

x / Contents

Localization, Biosynthesis, and Turnover of NADH-Cytochrome b 5 Reductase, a Protein That Exists in a Membrane-Bound and a Water-Soluble Form NicaBorgese 479 The Role of Receptor-Mediated Endocytosis in Iron Metabolism Yves-Jacques Schneider, Joseph N. Limet, Jean-Noel Octave, Ciaire Otte-Slachmuylder, Robert R. Crichton, and AndreTrouet 495

SECTION VI. ORGANIZATION AND TRANSPORT BY EPITHELIA IN CULTURE

Asymmetry in Epithelial Cells: Is the Tight Junction a Barrier to Lateral Diffusion in the Plasma Membrane? RR. Dragsten, J.S. Handler, and R. Blumenthal 525 Biosynthesis of the Catalytic Subunit of (Na+, K+)-ATPase in Toad Kidney and Toad Bladder Epithelial Cells K. Geering, M. Girardet, C. Bron, J.-P. Kraehenbühl, and B.C. Rossier 537 A Role for Calmodulin in the Activation of Adenylate Cyclase by Vasopressin Dennis A.Ausiello 543 Development of Na+-Dependent Hexose Transport In Vitro John S. Cook, Kurt Amsler, Ellen R. Weiss, and Carolyn Shaffer 551 Studies on Growth Regulation and the Mechanism of Transformation of the Kidney Epithelial Cell Line, MDCK: Importance of Transport Function to Growth Milton H. Saier, Jr., Serge Erlinger, and Paula Boerner 569 The Entry of Enveloped Viruses Into an Epithelial Cell Line Karl S. Matlin, Hubert Reggio, Ari Helenius, and Kai Simons 599 Index 613

Contributors

Kurt Amsler [551] Department of Human Genetics, Yale University School of Medicine, 333 Cedar Street, New Häven, CT 06510

Paul H. Atkinson [445] Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

Dennis A. Ausiello [543] Renal Unit, Massachusetts General Hospital, Fruit Street, Boston, MA 02114

Heinz Baumann [459] Department of Cell and Tumor Biology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263

R. Blumenthal [525] Section on Membrane Structure and Function, LTB, DCBD, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205

Paula Boerner [569] Department of Biology, University of California at San Diego, La Jolla, CA 92093

Liana Bolis [xix] Department of General Physiology, University of Messina, Sicily, Italy

J. Boonstra [211] Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Nica Borgese [479] C.N.R. Center of Cytopharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy

C. Bron [537] Institut de Biochimie, Universite de Lausanne, CH-1066 Epalinges, Switzerland

S.H. Bryant [321] Department of Pharmacology and Cell Biophysics, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, OH 45267

The boldface number in brackets following each contributor's name indicatestheopening page number of the author's article.

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xii / Contributors

Steven J. Bürden [239] Department of Anatomy, Harvard Medical Schoo!, 25 Shattuck Street, Boston, MA02115

K.L. Caldwell [373] School of Medicine, Washington University, St. Louis, MO 63130

D. Conte Camerino [321] Department of Pharmacolgy, Istituto di Farmacologia, Piazza G. Cesare, Policlinico, Bari, Italy

Daniele Carre [185] Equipe de Recherche Biologie du Developement, Station Zoologique, 06230 Villefranche-sur-Mer, France

M. Chambard [403] Universite d'Aix-Marseille, Marseilles, France

Patrick Chang [185] Salzburg University, Salzburg, Austria

John S. Cook [71,551] Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830

Jacky Cosson [185] Equipe de Recherche Biologie du Developement, Station Zoologique, 06230 Villefranche-sur-Mer, France

Marie Paule Cosson [185] Equipe de Recherche Biologie du Developement, Station Zoologique, 06230 Villefranche-sur-Mer, France

Evelyne Coudrier [89] Department of Cell Biology, EMBL, Postfach 10.2209, D-6900 Heidelberg, Federal Republic of Germany

Robert. R. Crichton [495] Universit6 Catholique de Louvain, International Institute of Cellular and Molecular Pathology, Brüssels, Belgium

S.W. de Latt[211] Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Umberto di Porzio [337] Institute of Molecular Embryology C.N.R., Via Toiano 2, 80072 Arco Feiice, Naples, Italy

Bernhard Dobberstein [23] European Molecular Biology Laboratory, Heidelberg, Germany

Darreil Doyle [459] Department of Cell and Tumor Biology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263

P.R. Dragsten [525] Procter and Gamble Company, Box 39175, Cincinnati, OH 45247

F. Dube [139] Sciences Biologiques, Universite de Montreal, CP 6128 Montreal PQ, Canada

Contributors / xiii

Peter Ekblom [429] Department of Pathology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland

Scott D. Emr [3] Department of Biochemistry, University of California, Berkeley, CA 94720

David Epel [171] Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, CA 93950

Serge Erlinger [569] Unite de Recherches de Physiopathologie Hepatique, Höpital Beaujon, F-92118, Clichy Cedex, France

Mariela Estenoz [337] Institute of Molecular Embryology C.N.R., Via Toiano 2, 80072 Arco Feiice, Naples, Italy

Douglas M. Fambrough [259] Department of Embryology, Carnegie Institute of Washington, Baltimore, Maryland 21210

C J . Feo [121] Institut de Pathologie Cellulaire, I.N.S.E.R.M. Unite 48, Höpital de Bicötre, 78 rue du G6n6ral Ledere, 94270 Kremlin Bic£tre, France

Giovanna Ferrari [311] Department of Cytopharmacology, Fidia Research Laboratories, Via Ponte della Frabbrica 3/A, 35031 AbanoTerme, Italy

B.M.J. Foxwell [59] Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom

J. Gabrion [403] Centre de Recherches de Biochimie des MacromolGcules, Montpellier, France

Gerald E. Gaull [349] Departments of Human Development and Nutrition and Pathological Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314

K. Geering [537] Institut de Pharmacologie, Universite de Lausanne, Bugnon 21, CH-1011 Lausanne, Switzerland

Gerhard H. Giebisch [xix] Department of Physiology, Yale University, New Häven, CT 06520

Jean Pierre Girard [185] Physiologie Cellulaire, Universite de Nice, 06000 Nice, France

M. Girardet [537] Institut de Biochimie, Universite de Lausanne, CH-1066 Epalinges, Switzerland

L. Glaser [373] School of Medicine, Washington University, St. Louis, MO 63130

xiv / Contributors

Alfredo Gorio [299, 311] Department of Cytopharmacology, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy

P. Guerrier [139] Station Biologique, Place Georges Teissier, 29211 Roseoff, France

John Hakimi [445] Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

J.S. Handler [525] NHLBI, Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health, Building 10, Room 6N30, Bethesda, MD 20205

M.T. Häuptle [389] Institut de Biochimie, Universite de Lausanne, CH-1066 Epalinges, Switzerland

Ari Helenius [599] Division of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Häven, CT 06510

Bernd Hennig [37] Institut für Biochemie, Universität Göttingen, Humboldtallee 7, 3400 Göttingen, Federal Republic of Germany

F. Herbst [107] Max-Planck-Institut für Biophysik, Frankfurt, Germany

G. M. Hillman [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Joseph F. Hoffman [xix] Department of Physiology, Yale University, New Häven, CT 06520

J.D. Jamieson [413] Section of Cell Biology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Häven, CT 06510

Manfred L. Karnovsky [329] Harvard Medical School, Harvard University, Cambridge, MA 02138

J.-P. Kraehenbühl [389, 537] Institut de Biochimie, Universite de Lausanne, CH-1066 Epalinges, Switzerland

Sune Kvist [23] European Molecular Biology Laboratory, Heidelberg, Germany

I. Lax [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Alberta Leon [311] Department of Biochemistry, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy

A. Levi [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Contributors / xv

T.A. Libermann [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Joseph N. Limet [495] Universite Catholique de Louvain, International Institute of Cellular and Molecular Pathology, Brüssels, Belgium

Daniel Louvard [89] Department of Cell Biology, EMBL, Postfach 10.2209, D-6900 Heidelberg, Federal Republic of Germany

James L. Maller [157] Department of Pharmacology, University of Colorado School of Medicine, Denver, CO 80262

Karl S. Matlin [599] Department of Cell Biology, European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, Federal Republic of Germany

J. Mauchamp [403] Universite d'Aix-Marseille, Marseilles, France

G. Mazzei [139] Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322

L Meijer [139] Station Biologique, Place Georges Teissier, 29211 Roseoff, France

David I. Meyer [23] European Molecular Biology Laboratory, Heldelberg, Germany

D. Mitolo-Chieppa [321] Deparment of Pharmacology, Istituto di Farmacologia, Piazza G. Cesare, Policlinico, Bari, Italy

W.H. Moolenaar[211] Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

M. Moreau [139] Station Biologique, Place Georges Teissier, 29211 Roseoff, France

C.L. Mummery [211] Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Walter Neupert [37] Institut für Biochemie, Universität Göttingen, Humboldtallee 7, 3400 Göttingen, Federal Republic of Germany

M.G. Nunzi [299] Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy

Richard A.D. O'Brien [247] Department of Anatomy and Embryology, Centre For Neuroscience, University College London, Gower Street, London WC1E 6BT, England

Jean-Noel Octave [495] Universite Catholique de Louvain, International Institute of Cellular and Molecular Pathology, Brüssels, Belgium

xvi / Contributors

Anna J.C. Östberg [247] Department of Anatomy and Embryology, Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, England

Robert E. Oswald [287] Department of Pharmacology, Cornell University, New York State College of Veterinary Medicine, Ithaca, NY 14853

Ciaire Otte-Slachmuylder [495] Universite Catholique de Louvain, International Institute of Cellular and Molecular Pathology, Brüssels, Belgium

R. Passing [107] Max-Planck-Institut für Biophysik, Frankfurt, Germany

Patrick Payan [185] Physiologie Cellulaire, Universite de Nice, 06000 Nice, France

George Perry [171] Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030

P. Polato [299] Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy

LR. Pollack [71] Department of Developmental Biology and Cancer, Albert Einstein School of Medicine, Bronx, NY 10461

L. Racine [389] Institut de Biochimie, Universite de Lausanne, CH-1066 Epalinges, Switzerland

Linda L. Randall [15] Biochemistry-Biophysics Program, Washington State University, Pullman, WA 99164

Graeme A. Reid [49] Department of Biochemistry, Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056, Basel, Switzerland

Hubert Reggio [89,599] Department of Cell Biology, European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, Federal Republic of Germany

B.C. Rossier [537] Institut de Pharmacologie, Universite de Lausanne, Bugnon 21, CH-1011 Lausanne, Switzerland

Richard L. Rotundo [259] Department of Embryology, Carnegie Institute of Washington, Baltimore, Maryland 21210

V. Rudioff [107] Max-Planck-Institut für Biophysik, Frankfurt, Germany

Milton H. Saier, Jr. [569] Department of Biology, University of California at San Diego, La Jolla, CA 92093

Christian Sardet [185] Groupe de Biologie Marine de CEA, Station Zoologique, 06230 Villefranche-sur-Mer, France

Contributors / xvii

Lauri Saxen [429] Department of Pathology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland

Gottfried Schatz [49] Department of Biochemistry, Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056, Basel, Switzerland

J. Schiessinger [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Manfred Schleyer [37] Institut für Biochemie, Universität Göttingen, Humboldtallee 7, 3400 Göttingen, Federal Republic of Germany

Bernd Schmidt [37] Institut für Biochemie, Universität Göttingen, Humboldtallee 7, 3400 Göttingen, Federal Republic of Germany

Tobias Schmidt [171] Zoologisches Institut, Universität München, Luisenstrasse 14, 800 München 2, West Germany

Yves-Jacques Schneider [495] Laboratoire de Chimie Physiologique, International Institute of Cellular and Molecular Pathology, B 1200 Bruxelles, Belgium

A.B. Schreiber [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

D. Schubert [107] Max-Planck-Institut für Biophysik, Frankfurt, Germany

Carolyn Shaffer [551] Biology Division, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, TN 37830

Thomas J. Silhavy [3] Cancer Biology Program, Frederick Cancer Research Facility, Frederick, MD 21701

Kai Simons [599] Department of Cell Biology, European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, Federal Republic of Germany

John A. Sturman [349] Departments of Human Development and Nutrition and Pathological Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314

Y. Suard [389] Institut de Biochemie, Universite de Lausanne, CH-1066 Epalinges, Switzerland

M.J.A. Tanner [59] Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom

E. H. Täte [71] Los Alamos Scientific Laboratory, Los Alamos, NM 87544

xviii / Contributors

Martin Teintze [37] Institut für Biochemie, Universität Göttingen, Humboldtalle 7, 3400 Göttingen, Federal Republic of Germany

Rupert Timpl [429] Max-Planck-Institut für Biochemie, Martinsried bei München, D-8033, Federal Republic of Germany

Gino Toffano [311] Department of Biochemistry, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy

Andre Trouet [495] Universite Catholique de Louvain, International Institute of Cellular and Molecular Pathology, Brüssels, Belgium

P.T. van der Saag [211] Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

E.J.J. van Zoelen [211] Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

B. Verrier [403] Universite d'Aix-Marseille, Marseilles, France

J.P. Vilain [139] Biologie Animale, Universite de Lille 1, BP 36, 59650 Villeneuve d'Ascq, France

Gerta Vrbovä [247] Department of Anatomy and Embryology, Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, England

Ellen R. Weiss [551] University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, TN 37830

Guang Y. Wen [349] Departments of Human Development and Nutrition and Pathological Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314

Henryk M. Wisniewski [349] Departments of Human Development and Nutrition and Pathological Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314

Y. Yarden [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Y. Yashuv-Gan [359] Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

R. Zanoni [299] Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy

Michael A. Zoccoli [329] School of Medicine, Harvard University, Cambridge, MA 02138

Preface The central role that membranes play in cellular processes is becoming in-

creasingly apparent. The present Symposium contains contributions on vari-ous aspects of involvement of membranes in growth and development. These include genetic determinants and the synthesis and assembly of membrane constituents such as transport proteins and receptors during cell differentia-tion and cell growth. Neuromuscular and epithelial development together with intracellular organelles are considered. The interplay between various mem­brane Systems and their modulation by cellular control mechanisms are a con-stant theme of the various chapters. Each contributor considers the historical perspectives and the current Status of that particular field. The subjects are timely, and it is hoped that others will be stimulated by the new concepts of this emerging field. It should also be mentioned that the contributions in this volume were presented at the biennial International Conference on Biological Membranes held at Crans-sur-Sierre, Switzerland, June 15-19, 1981.

J.F. Hoffman G.H. Giebisch

L. Bous

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Membranes in Growth and Development, pages 37-47 © 1982 Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY 10011

Biogenesis of Mitochondrial Membrane Proteins Martin Teintze, Bernd Hennig, Manfred Schleyer, Bernd Schmidt, and Walter Neupert

INTRODUCTION

Eukaryotic cells, in contrast to prokaryotic cells, are divided into a number of compartments. By creating these compartments (ie, organelles), the eukary­otic cell gains a host of new properties and capabilities. The presence of specif­ic reaction vessels enclosed by membranes within the cell allows, for instance, that metabolic pathways can be used simultaneously in opposite directions or that substances (such as Ca+ +) can be sequestered within the cell and released when needed to initiate reactions. Ion or proton gradients can also be gener-ated across the membranes of the organelles to drive the synthesis of sub­stances such as ATP. However, this increase in capabilities brings with it a se-ries of new problems for the eukaryotic cell. A major one is that the proteins of the intracellular organelles are, with few exceptions, synthesized on cytoplas-mic ribosomes [Chua and Schmidt, 1979]. They must be transported into the organelles during or after translation. This immediately raises a series of ques-tions. The membranes of the organelles are impermeable to most low molecu­lar weight Compounds and practically completely impermeable to macromole-cules such as proteins. On the other hand, cytoplasmic ribosomes synthesize proteins for a number of different organelles in the cell. The cell must there-fore have mechanisms that specifically insert newly synthesized proteins into the proper organeile.

An explanation of the molecular mechanisms of intracellular protein trans-port can only be achieved if Single, well-defined proteins that can be assigned to one particular cell compartment are followed over the entire path from the synthetic origin to the functional site. This requires the isolation and purifica-tion of these proteins and the preparation of specific antibodies, so that the very small quantities of precursors or intermediates involved in the transfer process can be found.

38 / Teintze et al

POST-TRANSLATIONAL TRANSFER OF PROTEINS INTO MITOCHONDRIA

The intracellular transport of quite a few proteins into mitochondria and chloroplasts has been studied in this manner [Chua and Schmidt, 1979; Neu-pert and Schatz, 1981]. Some work has also been done recently on glyoxyso-mal and peroxisomal proteins [Zimmermann and Neupert, 1980; Frevert et al, 1980; Roberts and Lord, 1981; Goldman and Blobel, 1978; Lazarow, 1980]. The results have shown that the transport into these organelles is posttransla-tional [Hallermeyer et al, 1977; Harmey et al, 1977; Korb and Neupert, 1978; Maccechini et al, 1979; Raymond and Shore, 1979; Morita et al, 1981; Conboy and Rosenberg, 1981]. This is in contrast to the cotranslational transfer that takes place in the endoplasmic reticulum (ER) to insert proteins into ER mem­branes and into plasma membrane, and to transfer secretory proteins into the inner of the ER [Palade, 1975; Blobel and Dobberstein, 1975].

There is not one unique mechanism by which proteins are inserted into mi­tochondria after synthesis on free ribosomes; different proteins, even if they are subunits of the same enzyme complex, will often use a somewhat different mechanism to insert into the mitochondria. There are, however, several unify-ing features of the transport pathway that have been observed. First of all, ev-ery protein that has been studied thus far is synthesized as a precursor that is in some way different from the mature functional protein. This is of course a necessary feature, both for membrane proteins, which must change from a form soluble in the cytosol to one soluble in the membrane, and for proteins solubilized inside the organeile, which must be irreversibly altered to prevent them from leaving their compartment. IMPORT OF PROTEINS INTO MITOCHONDRIA OCCURS WITH AND WITHOUT PROTEOLYTIC PROCESSING

Many of the proteins whose transport has been studied are synthesized as larger precursors. Their apparent molecular weights exceed those of the ma­ture proteins by anywhere from 500 daltons for subunit VII of the Neurospora cytochrome bd complex to 6,000 daltons for cytochrome d and some of the mitochondrial ATPase subunits. This additional sequence is then removed by a proteolytic enzyme during or after the transfer process. This form of post-translational transfer with proteolytic processing has been observed, for in-stance, for ribulose-l,5-bisphosphate carboxylase in chloroplasts [Dobber­stein et al, 1977] and for subunit 9 of the mitochondrial oligomycin sensitive ATPase (OS-ATPase) [Michel et al, 1979; Zimmermann et al, 1981] as well as many of the subunits of the cytochrome bd complex [Nelson and Schatz, 1979; Cote et al, 1979] (Teintze et al, unpublished) and subunits of cytochrome oxidase [Lewin et al, 1980; Mihara and Blobel, 1980; Schmelzer and Heinrich,

Biogenesis of Mitochondrial Membranes / 39

1980]. In the case of cytochrome d, the proteolytic processing seems to take place in two separate Steps via a form of intermediate molecular weight that is already located somewhere within the mitochondria (Teintze et al, unpub-lished; Ohashi and Schatz, personal communication). On the other hand, sub­unit VI of the cytochrome bd complex appears not to be synthesized as a lar-ger precursor (Teintze et al, unpublished).

For proteins that do not have a precursor with a larger molecular weight such as cytochrome c, ADP/ATP carrier, and mitochondrial porin [Korb and Neupert, 1978; Zimmermann et al, 1979a,b] (Freitag and Neupert, unpub­lished), something eise must trigger a conformational change. In the most ex-tensively studied example, cytochrome c, it is the attachment of the heme group to apocytochrome c that results in a conformational change when the protein transverses the outer mitochondrial membrane [Hennig and Neupert, 1981] (Fig. 1). The fact that apocytochrome c changes its conformation upon attachment of the heme group to form holocytochrome c is demonstrated by the absence of cross-reactivity between antibodies prepared against apo- and holocytochrome c in our laboratory [Korb and Neupert, 1978] and by the fact that cold excess holocytochrome c cannot compete with radioactive apocyto­chrome c for binding to and transfer into Neurospora mitochondria [Hennig and Neupert, 1981]. In the cases of the ADP/ATP carrier and ATPase subunit 9, the precursors synthesized in vitro in a heterologous cell-free system were found to be present as soluble aggregates with molecular weights in the ränge of 100,000 to 500,000 [Zimmermann and Neupert, 1980b] (Schmidt and Neu­pert, unpublished). The mature proteins are soluble only in the presence of de-tergents and cannot be used to inhibit the transfer of the precursors into mitochondria.

MUTUAL RECOGNITION OF PRECURSORS AND ORGANELLES IS MEDIATED BY RECEPTORS ON THE MITOCHONDRIAL SURFACE

The precursor proteins, which are synthesized on free cytoplasmic ribo­somes, must have a method of recognizing the organeile for which they are in-tended and a method of entering into or transversing a membrane which is normally impermeable to proteins. The most logical mechanism for such a process is the presence of a specific receptor in the outer membrane of the or­ganeile. There is, in fact, considerable evidence to support the existence of such receptors (although none have been isolated thus far). Neurospora pro­teins can be synthesized in vitro and the postribosomal supernatant incubated with Neurospora mitochondria under conditions where the precursors to the mitochondrial proteins are transferred into the mitochondria. When the mito­chondria are then reisolated, washed, and lysed, and antibodies to a specific protein are added, precursors bound to the mitochondria are immunoprecipi-

40 / Teintze et al

OM IM

Holocytochrome _c

Fig. 1. A possible mechanism for the assembly of cytochrome c. Apocytochrome c is synthe­sized on free cytoplasmic ribosomes and then bound to a receptor in the outer mitochondrial membrane in such a way that the heme group can be attached by a heme lyase in the intermem-brane space. Attachment of the heme group causes a conformational change that completes the transfer and results in mature, functional holocytochrome c.

Biogenesis of Mitochondrial Membranes / 41

tated along with the mature protein (Fig. 2). If the mitochondria are treated with Proteinase K prior to lysis, the precursors are degraded whereas the ma­ture proteins are not. The preferential sensitivity of precursor proteins to added proteases enables one to distinguish the bound precursor from the ma­ture protein when both have the same molecular weight, as in the case of the ADP/ATP carrier. If the transfer in the reconstituted system is blocked by the presence of energy inhibitors such as valinomycin and carbonyl Cyanide m-chlorophenyl hydrazone (CCCP), only the bound precursor is found [Zim­mermann et al, 1981].

This suggests that the precursors are bound by some type of receptor in the outer membrane and that the transfer is energy-dependent, whereas the bind-ing of the precursor is not. It is possible that in vivo a precursor may be recog-nized by its receptor before synthesis is completed [Ades and Butow, 1980], but the transfer appears to be always posttranslational. In the case of cyto­chrome c, the presence of specific saturable binding sites can be shown because excess unlabeled apocytochrome c, but not holocytochrome c, will compete with labeled apocytochrome c and inhibit its transfer into the mitochondria [Hennig and Neupert, 1981]. The transfer of apocytochrome c does not seem to be energy-dependent, but it can be inhibited by deuterohemin, which cannot be covalently attached to the protein because it lacks the vinyl groups of the natural prosthetic group protoheme. In the presence of excess deuterohemin, bound apocytochrome c accumulates on the mitochondria. If these are then reisolated and resuspended in a medium containing protohemin, but no addi-tional precursor, the bound apocytochrome c is transferred into the mitochon­dria and converted to holocytochrome c [Hennig and Neupert, 1981] (Table I).

THE TRANSPORT OF MANY PROTEINS INTO MITOCHONDRIA IS DEPENDENT ON THE MEMBRANE POTENTIAL

The import of many cytoplasmically synthesized proteins into mitochon­dria is energy-dependent. This was shown for the ADP/ATP carrier and sub­unit 9 of the OS-ATPase [Zimmermann et al, 1981] and for cytochrome d and subunit V of the cytochrome bei complex (Teintze et al, unpublished) in in vi­tro transfer experiments using Neurospora mitochondria. In addition, the ener­gy dependence of the transfer of subunits a and ß of FrATPase, cytochrome d, and subunit V of the cytochrome bd complex has been investigated using pulse-chase experiments in vivo with yeast cells, and it was concluded that pro-cessing of the precursor proteins was dependent on the ATP level in the mito­chondria, rather than on the membrane potential [Nelson and Schatz, 1979]. All these proteins are located within or on the matrix side of the inner mito­chondrial membrane. The energy dependence of the transfer of the ADP/ATP

42 / Teintze et al

Transfer in vitro of b c r Complex Subunits

Subunit I Sup. Mit.

Subunit I Sup. Mit.

Cytochrome c f

Sup Mit.

Biogenesis of Mitochondrial Membranes / 43

TABLE I. Binding of Apocytochrome c to Receptor Sites on Mitochondria8

Cytochrome c bound to mitochondria Counts/min per % Total 3H-cyto-

mitochondrial protein chrome c of control Apocyto- Holocyto- Apocyto- Holocyto-

Additions chrome c chrome c chrome c chrome c

None (control) 682 4168 14 86 4- Deuterohemin (10 nmol/ml) 4330 1185 89 24 Deuterohemin (10 nmol/ml), then

mitochondria washed with 0.44 M sucrose, 1 mM EDTA, 10 mM Tris/HCl, pH 7.4 3752 1120 77 23

+ Deuterohemin (10 nmol/ml), then incubation with protohemin (10 nmol/ml) 1917 2934 40 60

+ Deuterohemin (10 nmol/ml), then incubation with apocytochrome c of Neurospora crassa (10 nmol/ml) 710 1160 15 24

4- Deuterohemin (10 nmol/ml), then incubation with holocytochrome c of Neurospora crassa (10 nmol/ml) 3742 1200 77 25

aApocytochrome c was synthesized in a cell-free System of Neurospora crassa in the presence of 3H-leucine. A postribosomal supernatant was prepared which contained the 3H-labeled apocy­tochrome c. Mitochondria were isolated from Neurospora cells and incubated with the postribo­somal supernatant in the absence or presence of deuterohemin for 15 min at 25°C. Then mitochondria were reisolated. When indicated they were resuspended in fresh unlabeled postri­bosomal supernatant. Then protohemin, apocytochrome c, or holocytochrome c was added and incubation was continued for 15 min. Mitochondria were then reisolated. From one-half of each of the various mitochondrial samples apocytochrome c was immunoprecipitated, and from the other half holocytochrome c, employing specific antibodies. Immunoprecipitates were analyzed by SDS gel electrophoresis, and radioactivity in the cytochrome c peak was determined. The sum of the radioactivities in apocytochrome c and holocytochrome c in the sample without any addi-tion was set at 100%.

Fig. 2. Transfer in vitro and proteolytic processing of cytochrome bc, complex subunits. Ra-dioactive precursors to cytochrome bc, complex subunits were synthesized in a reticulocyte lysate cell-free System, and the postribosomal supernatant was incubated with Neurospora mito­chondria for 1 h at 25°C. The mitochondria were then separated from the supernatant by centri-fugation and both fractions were adjusted to 1 °7o Triton, 0.3 M NaCl. Immunoprecipitation was then carried out using antibodies against the individual subunits, foilowed by SDS gel electro­phoresis and autoradiography. The antibodies precipitated both the precursors (open arrows) and the mature proteins (black arrows); the arrow labeled I points to the intermediate form of cytochrome c, (see text).

44 / Teintze et al

carrier (Fig. 3) and of subunit 9 of the OS-ATPase was studied in detail in a reconstituted System employing isolated Neurospora mitochondria and the postribosomal supernatant of a rabbit reticulocyte lysate incubated with Neu­rospora RNA and (35S)methionine (Schleyer et al, unpublished). In these ex­periments, the uncoupler CCCP and the ionophore valinomycin (which break

Effect of various inhibitors on transfer of ADP/ATP carrier in vitro

< E 8>

E .8»

< o o

2E 3* o 8 »—

<

Q- a. _ o o .£ o o y o o P * o Q. Q. Ol < < O

Q_ O U U c o u

-Proteinase K -Proteinase K

»Proteinase K «Proteinase K

Fig. 3. The transport of the ADP/ATP carrier is dependent on the membrane potential. La­beled precursors were synthesized in a reticulocyte lysate cell-free system, and the postribosomal supernatant was incubated with Neurospora mitochondria for 1 h at 25 °C. The mitochondria were reisolated and then lysed in 1% Triton, 0.3 M KCl. The ADP/ATP carrier was immunopre-eipitated, electrophoresed on SDS gels, and autoradiographed. One or more of the following were present during the incubation when indicated: 2.6 mM ATP, 2.4 fM carboxyatraetyloside (CAT), 12.5/iMCCCP, 5/iMoligomycin. ProteinaseK(1 mg/ml, 30min, 25°C)wasused todi­gest the precursor bound to the mitochondria. The mature protein after transfer is protease-resistant.

Biogenesis of Mitochondrial Membranes / 45

down the membrane potential) inhibited the transfer of these proteins into the mitochondria. When oligomycin (which blocks the OS-ATPase) was used to-gether with valinomycin to ensure that the ATP in the mitochondria was not being hydrolyzed, the transfer was still blocked. CCCP together with oligomy­cin and ATP also had the same effect, showing that even when ATP is present at a high level in the mitochondrial matrix, the transfer will not take place in the absence of a membrane potential [Heidt et al, 1972]. Carboxyatractyloside (which blocks the ADP/ATP carrier) together with oligomycin cannot inhibit the transfer, although the mitochondria should be depleted of ATP [Klingen­berg, 1976]. This confirms that it is the membrane potential, rather than ATP, that is required for the import of the proteins into the mitochondria. Cyto­chrome c, located in the intermembrane space on the cytoplasmic side of the inner membrane, and the outer membrane porin both do not seem to require energy for their import [Zimmermann et al, 1981] (Freitag and Neupert, un­published). This is to be expected, since they are located outside the potential across the inner membrane.

TRANSFER OF NEUROSPORA PROTEINS INTO RAT LIVER MITOCHONDRIA

When labeled precursors to Neurospora proteins (in the postribosomal su­pernatant of a rabbit reticulocyte lysate incubated with Neurospora RNA) were incubated with isolated rat liver mitochondria, the results were similar to those obtained with Neurospora mitochondria. The precursors of the ADP/ ATP carrier and the mitochondrial porin were transferred to a protease-resis-tant location in the mitochondria (Freitag and Neupert, unpublished; Schleyer et al, unpublished). The precursor of subunit 9 of the OS-ATPase and subunits I and V of the cytochrome bd complex were processed to the molecular weights of the mature proteins (Teintze et al, unpublished; Schleyer et al, un­published); the precursor to cytochrome c, seems to have been processed only to the molecular weight of the intermediate form (see above) (Teintze et al, unpublished).

The energy dependence of the import into rat liver mitochondria also ap-peared to be similar to that in Neurospora. These results indicate that the structures of the receptors and proteases involved in the transfer of proteins in­to mitochondria, as well as the proteins themselves, must be highly conserved.

CONCLUSION In summary, proteins appear to be transported into mitochondria by the

following mechanism. First, the protein is synthesized on free cytoplasmic ri­bosomes as a precursor with or without an additional sequence, but definitely

46 / Teintze et al

with a conformation different from that of the mature functional protein. Next, the precursor protein is bound to a receptor on the outer mitochondrial membrane. The protein is then translocated across the membrane into the in-termembrane space, or possibly into the inner membrane or the matrix by way of a contact site between the two membranes. If the protein enters or crosses the inner mitochondrial membrane, this process is dependent upon the potential across this membrane and may also involve an additional protein in the mem­brane to catalyze the translocation. Düring or after the transfer from the outer membrane receptor to the functional site, any additional sequences are re-moved from the precursor and prosthetic groups are attached, if required. The components of this mechanism seem to be common to mitochondria from spe-cies as varied as yeast, Neurospora, and rat.

ACKNOWLEDGMENTS We are grateful to P. Knoch, S. Knill, and L. Sander for expert technical

assistance. This work was supported by the Deutsche Forschungsgemeinschaft Ne 101/17 and by the Fonds der Chemischen Industrie.

REFERENCES Ades I, Butow RA (1980): J Biol Chem 255:9925-9935. Blobel G, Dobberstein B (1975): J Cell Biol 67:835-851. Chua N-H, Schmidt GW (1979): J Cell Biol 81:461-483. Conboy, JG, Rosenberg LE (1981): Proc Natl Acad Sei USA 78:3073-3077. Cote C, Solioz M, Schatz G (1979): J Biol Chem 254:1437-1439. Dobberstein B, Blobel G, Chua N-H (1977): Proc Natl Acad Sei USA 74:1082-1085. Frevert J, Koller W, Kindl H (1980): Z Physiol Chemie 361:1557-1565. Goldman BM, Blobel G (1978): Proc Natl Acad Sei USA 75:5066-5077. Hallermayer G, Zimmermann R, Neupert W (1977): Eur J Biochem 81:523-532. Harmey MA, Hallermayer G, Korb H, Neupert W (1977): Eur J Biochem 81:533-544. Heidt H-W, Klingenberg M, Milovancev M (1972): Eur J Biochem 30:434-440. Hennig B, Neupert W (1981): Eur J Biochem (in press). Klingenberg M (1976) In Martonosi AN (ed): "The Enzymes of Biological Membranes," Vol 3,

New York: Plenum, pp 383-438. Korb H, Neupert W (1978): Eur J Biochem 91:609-620. Lazarow PB (1980): Ann NY Acad Sei 344:293-303. Lewin A, Gregor I, Mason TL, Nelson N, Schatz G (1980): Proc Natl Acad Sei USA 77:

3998-4002. Maccechini M-L, Rudin Y, Blobel G, Schatz G (1979): Proc Natl Acad Sei USA 76:343-347. Michel R, Wächter E, Sebald W (1979): FEBS Lett 101:373-376. Mihara K, Blobel G (1980): Proc Natl Acad Sei USA 77:4160-4164. Morita T, Mori M, Tatibana M, Cohen P (1981): Biochem Biophys Res Commun (99)623-629. Nelson N, Schatz G (1979): Proc Natl Acad Sei USA 76:4365-4369. Neupert W, Schatz G (1981): Trends Biochem Sei 6:1-4. Palade GW (1975): Science 189:347-358.

Biogenesis of Mitochondrial Membranes / 47

Raymond Y, Shore GS (1979): J Biol Chem 254:9335-9338. Roberts LM, Lord JM (1981): Eur J Biochem 119:43-49. Schmelzer E, Heinrich PC (1980): J Biol Chem 255:7503-7506. Zimmermann R, Paluch U, Neupert W (1979a): FEBS Lett 108:141-146. Zimmermann R, Paluch U, Sprinzl M, Neupert W (1979b): Eur J Biochem 99:247-252. Zimmermann R, Neupert W (1980a): Eur J Biochem 112:225-233. Zimmermann R, Neupert W (1980b): Eur J Biochem 109:217-229. Zimmermann R, Hennig B, Neupert W (1981): Eur J Biochem 116:455-460.

Index

Acetylcholine and nerve reinnervation, 300-301 and pharmacologic nerve block,

323-325 receptors, 239-244, 338

and calcium, 252-253, 256 in cultured muscle, 259-264,

278-284 and growth regulation, 378,

383, 388 immunologic study of, 239-244 membrane localization of, 287-

294 Acetylcholinesterase, 239-244, 259,

264-284, 303, 338 Acrosomal process, 192-194 Actin, 122, 191-192 a-Actinin, 90-97 Adenosine monophosphate (AMP)

cyclic (cAMP), 553, 555 in amphibian oocytes, 157-159,

167 and calcium, in meiosis, 143,

148-150 dibutyryl, 312-316, 377, 578 in erythrocyte membrane, 125,

127 and polarization in cultured thy-

roid cells, 404-407 and transport function, 578-579,

590-593 and Vasopressin, 543-549 and growth regulation, 374

Adenosine triphosphate (ATP), 19, 74-75, 329

and biogenesis of mitochondrial membrane proteins, 37-45

and calcium, 139, 189 and growth regulation, 374 (Na + , K +)ATPase, 313, 559, 600

and epidermal growth factor (EGF), 367, 371

in HeLa cells, 71-85 in neuroblastoma cells, 211,

227-234 and polarity in epithelial cells,

89-103 in toad,537-542 and transport function, 573, 585-

588, 592-595 Adenylate cyclase, 148-150, 157-167,

403, 543-549 Aequrin, 176-177 Alcoholism and iron overload, 495 Alkaline Phosphatase, 17-18 Alkalinization, cytoplasmic, at fertiliza­

tion, 179 Amantadine, 603 Ambystoma, 146-147 Amiloride, 231-234 Aminergic neurone development, 339-

343 Amino acid transport in MDCK and T,

cells, 590-592 a-Aminoisobutryic acid (AIB), 556-

558, 591 Aminopeptidase, 89-103, 600 Ammonia, role in egg activation, 202 Ammonium Chloride, 603-605 Amphibians, 140-145, 148

613

614 / Index

Anemia and iron overload, 495 hemolytic (human), 121-133 phenylhydrazine-induced, 61, 64-67

Angiogenesis, 438 Anion transport protein, 59-69 Ankyrin, 113, 122 Antibodies, monoclonal

against synaptic components, 239-244

vs EGF receptor, 362-366 Antigen(s)

H-2, mouse, 29 HLA-DR, assembly of, 27-34 histocompatibility, 480

Arabinose, 17-20 Arachidonate, 177-178 Assembly

of HLA-DR antigens, 27-34 membrane protein, 23-34 see a l s o Self-assembly

Assymetry, epithelial cell, 525-535 5-Azido-trimethisoquin (5AT), 293

Bacteria, signal hypothesis of protein transport in, 3-13

Barnea, 140-142 Bindin, 192 Brain, development, mammalian, 337-

345 Brain metabolism, slow-wave sleep,

329-334 Bufo marinus, 538 Bumetamide, 585 a-Bungarotoxin, 240, 261-264, 277-

281, 383

Calcium intracellular, in fertilization, 171-

181 ions, 214-216, 224-231, 278

in neuromuscular junction reor-ganization, 253, 256

in oocyte meiosis reimitation, 139-150

in muscle reinnervation, 300-301

release in fertilization, 176-177 required for acrosomal process,

192 role in egg activation, 197-199 triggering of development, 177 and Vasopressin, 543-549

see a l s o Salt transport Calf serum, fetal, 212-215 Calmodulin, 148-150, 543-549 Carbonyl Cyanide m-clorophenylhydra-

zone (CCCP), 18, 41, 45, 51-56, 278, 284

Carboxyatractyloside, 45 Carcinogenesis, 413, 417, 422-426. See

a l s o : Tumor promotors; Viruses, oncogenic

Carcinogens, 569-570 Carcinoma, 575

human (A-431), 359-371 pancreatic acinar cell, 422-426

Casein, 390, 392-400 Cat, taurine requirements in, 349-354 Catecholamine, 353 Cathepsin B, 508-509, 514 Cathepsin D, 479, 481,488 Cell cycle, neuroblastoma, 217-234 Cell line(s)

epithelial, entry of virus into, 599-609

fibroblast, human 1MR91, 376 kidney, dog MDCK, 89-103, 525-

535, 569-595 kidney, pig LLC-PK,, 525-

535, 543-549, 551-566 kidney, toad A6, 525-535 mouse 3T3, 374-377, 382 muscle-like BC3H1, 373-385 3T6, 571, 573 toad TBM and TB6c, 538-542

Cell(s) amino acid transport in MDCK and

T„ 590-592 Carcinoma, human epidermoid (A-

431), 359-371 CHO, 143-144 differentiation, neuronal, 311-317

Index / 615

epithelial, assymetry of, 525-535 epithelial, polarized, 89-103 fibroblasts, cultured rat, 497-501 growth regulation, 167 HeLa, 71-85, 143-144, 573 hepatic, rat, 459-476 hepatoma, cultured, 460-476 L-929, 469-474 neuroblastoma, 211-234, 312-315,

317 pheochromocytoma (PC12), 308,

315-317 photoreceptor, 352-353 red, and iron metabolism, 496 red, deformability, 124, 128-130 Reuber H-35, 469 Schwann, 376-377, 384 shape, and hormone responsiveness,

389-400 T„ 584-595 teratocarcinoma, mouse, 501 thyroid, cultured, polarization of,

403-411 Cell-cell contact, growth control by,

373-385 Cell-surface

component turnover, 71-85 mediated change, 171-181

Central nervous System (CNS), role of taurine in development, 349-354

Chloride cation, 584-595 role in egg activation, 201-206

Chloroquine, 498-504, 514-516, 603 Cholera toxin, 157-159, 314 Cholesterol, 578 Cholinesterase inhibitors, 253 Colchicine, 271, 278, 284, 321 Collagen, 292, 391-395, 414-416, 420-

424, 429-439 Concanavalin A, 61 Cortex, kidney, 89-103 Creatine kinase, 378-388 Cupule, egg, 186-191 Cyanogen bromide cleared EGF,

365-367, 370-371

Cycloheximide, 31, 50, 147, 273, 278, 379-385, 558

Cysteinesulfiric acid, 349-354 Cystine, 349, 353 Cytochalasin B, 563-564 Cytochrome, 50, 54 Cytochrome b5, 480-491 Cytokeratin, 241 Cytoplasmic alkalinization at fertiliza­

tion, 179 Desmin, 241 Deuterohemin, 41, 43 Development, calcium triggering of,

177 Dexamethasone, 393 Diazobenzene-sulfonate, 331 Dibutyryl cAMP, 312-316, 377 DIDS, 331-334 Differentiation

lobuloalveolar, 389-400 neuronal cell, 311-317

Digitalis-like drugs, 83 Diisopropyl fluorophosphate (DFP),

265-268, 272, 275-281 Dimethylmalic (DMM) anhydride, 107-

108, 113 Dinitrophenol, 18-19, 278, 284 Dipeptidyl peptidase, 489 Dog, kidney cell line MDCK, 89-103,

526-535, 569-595 Domain(s)

on sperm membrane, 194 trypsin-insensitive (TID), 289 surface and cytoplasmic, 89-103

Dopamine-/3-hydroxylase (BDIT), 340

Echinoderms, sperm binding in, 192-195

Eel, electric, 261, 264-265, 287-294 Egg(s)

activation by calcium influx, 171-173

activation by sperm in sea urchin, 185, 195-207

attraction to sperm in siphono-

616 / Index

phores, 185-195 fish, 171-172

Ektacytometry, 121-133 Elastase, 24-29 Electric eel (Electrophorus electricus),

261, 264-265, 287-294 Electric ray (Torpedo, Narcine), 240-

244, 274, 287-294 Electrical qualities of muscle mem­

brane, 321-326 Elliptocytosis, 121, 125, 131-133 Embryo(s)

sea urchin, gastrulation, 420 tissue interactions, 430

Endocytosis, receptor-mediated, and iron metabolism, 495-518

Endoplasmic reticulum (ER), 479-489 membranes, 329-334 rough (RER), 4, 261-262, 283 translocation of nascent peptides

across, 24-27 Endotoxins, 311 Energy requirements in protein trans­

port, 18-20, 41-46, 50 Enzyme, heme, 496; see a l s o specific

enzymes Epidermal growth factor (EGF), 212-

216, 359-371, 393-395 Epithelium

glandulär, 413-426 leaky, 551 tight vs leaky, 529

Epithelial cells assymetry, 525-535 polarized, 89-103

Erythrocyte membrane protein deficien-cy, 121-133

Erythroid cell differentiation, 60-61 Escherechia coli, 4-10, 15-20 17 ß Estradiol, 393 Exocrine glands, morphogenesis of,

414-421 Extracellular matrix (ECM), and kidney

differentiation, 429-439

F-actin, 122 Ferriheme, 506

Ferritin, 99-102, 241, 496-518 Fertilization

calcium release in, 176-177 cytoplasmic alkalinization at, 179 intracellular calcium in, 171-181 in marine invertebrates, 185-207 model for cell-surface mediated

change, 171-181 potassium in, 174-175

Fetal calf serum, 212-215 Fibroblasts, 374-376

human foreskin, 364-365 mouse, 459-476 rat, 459-476, 497-501

Fibronectin, 292, 416, 430, 432-439 Fish eggs, 171-172 Fluorescent lipid probes, 527-535 Fluoride, 159-160 Frog neuromuscular Synapse, 239-244 Furosemide, 585-587 G/F protein, 160-161 /3-Galactosidase, 4-5 Galactosyltransferase, 508-509 Gangliosides, 307-308, 311-317 Gene fusion in protein localization, 4 -

10 Germinal vesicle breakdown (GVBD),

139-150, 158-163, 166 Globin, 506 Glucagon, 578 Glucocorticoids, 339, 389-390 Glucose-6-phosphohydrolase, 329-334 Glycogen, 330-331 Glycophorin A, 480 Glycoproteins

plasmalemmal, 413-426 Tamm-Horsfall, 437 viral envelope, 480

Glycosaminoglycans, 414, 430 Golgi

apparatus, 79, 608 locus of ACh receptors, 261-262,

282-283 complex, 103, 479-489 region, 445

Growth control

Index / 617

by cell-cell contact, 373-385 regulation in MDCK cell line, 565-

595 and transport function, 569-595

Guanosine diphosphate (GDP), 159 Guanosine triphosphate (GTP), 159 Haptoglobin, 496 Heme enzymes, 496 Heme-hemopexin complexes, liver up-

take of, 505-506, 511-516 Hemoglobin-haptoglobin complexes,

liver uptake of, 505-511, 517 Hemoplexin, 496 Hemosiderin, 496 Hepatoma, cultured cells, 460-476 Hexamethylene bisacteamine (HMBA),

555-558 ß-Hexosaminidase, 81 Hexose transport, sodium-cation-

dependent, 551-566 Hormone(s), 311

and growth, 573 responsiveness, and cell shape, 389-

400 thyroid-stimulating, 403-410

Hydrocortisone, 390, 394-400, 578, 581, 592

Hydrogen, 215-216 cation, 584-595 role in egg activation, 200-206

Hyppocampus, 340

Immunoglobulin(s) IgA, 507, 510-511 IgM, 490-491

Immunologie identification of intra­cellular postsynaptic protein, 239-244

Innervation, polyneuronal, 247-256 Insulin, 82-83, 362

action on oocytes, 163-167 and cell shape in cultured mammary

cells, 389-390, 393-395 and growth regulation in MDCK cell

line, 573, 578-581, 592, 594 -like growth factor (IGF), 167

Interferon, 311 Intestinal mueosa, 89-103 Intramembrane particles (IMP), 220-

224 Inulin, 500 Invertebrates, marine, fertilization in,

185-207 Iodide, 409 Ions. See specific ions Iron

metabolism, and receptor mediated endocytosis, 495-518

overload, 495, 516 Isobutylmethylxanthine, 578 Isozymes, BB, rabbit, 387-388

Junction, tight, 525-535, 553, 575

Kidney cortex, 89-103 morphogenesis of, 431-439 see a l s o Cell line(s)

a-Lactalbumin, 390, 392-400 0-Lactamase, 17-18 Lactogen, placental, 390 Lactoperoxidase, 61-62 Lambda receptor, 3-13 Laminin, 292, 420, 424, 430-439 Lipids, and acetylcholine reeeptors,

287-294 Lipoxygenase, 177-178, 181 Liver

rat, 459-476, 479-491 uptake of heme-hemopexin com­

plexes, 505-506, 511-516 uptake of hemoglobin-haptoglobin

complexes, 505-511, 517 Lobuloalveolar differentiation, 389-400

Magnesium cation, 584-595 Maleyl, 107 Maltose, 3, 5, 16-20 Mammals, brain development, 337-345 Mammary gland, 389-400 Manganese, 52, 160, 584-595 Mannose, 445-455

618 / Index

Marine invertebrates, fertilization in, 185-207

Mechanism of calcium release at fertili­zation, 176-177

Meiosis, oocyte, 139-150 Membrane(s)

acetylcholine receptors, localization of, 287-294

erythrocyte, protein deficiency, 121-133

growth in neuroblastoma cell cycle, 217-234

inhibition of cell growth, 375 mitochondrial membrane, biogenesis

of, 37-46 muscle, electrical qualities of, 321 —

326 plasma

glycoconjugate metabolism, 459-476

lateral diffusion, 525-535 steroid-growth hormone interac-

tion with, 157-167 potential and permeability in neuro­

blastoma, 224-227 protein assembly, 23-34 see a l s o Endoplasmic reticulum,

Intramembrane particles Metabolism

brain, slow-wave sleep, 329-334 glycoconjugate, in plasma mem­

brane, 459-476 iron, and receptor mediated endo-

cytosis, 495-518 Metahemoglobin, 480-481 Methimazole (MMI), 407-410 Methionine, 349, 353, 593 Methylamine, 498-504, 514-516 ce-Methylaminoisobutryic acid (meAIB),

556 ot-Methylglucoside, 551-555, 559-565 Methylisobutyl xanthine (MIX), 555-

559, 562-565 Mitochondria, 479-489

membrane protein of, biogenesis of, 37-46

protein transport in, 49-58 rat liver, 45-46 surface receptors, 39 yeast, 49-58

Moliuscs, 140-142 Monensin, 186, 271, 278, 280-284 Monoclonal antibodies. See Antibodies,

monoclonal Morphogenesis

exocrine glands, 414-421 kidney, 431-439 pancreas, 413-426

Morphogenesis, nornal exocrine gland, 414-421

Mouse athymic nude, 569-575 fibroblasts, 459-476 myeloma (NSI), 363 teratocarcinoma cells, 501 Ii chains, 490

Mucosa, intestinal, 89-103 Muggiaea kochi, 188-191 Muscle(s)

cultured acetylcholine receptors in, 259-

264, 278-284 acetylcholinesterase in, 259,

264-284 EDL, 322-326 extensor digitorum longus, rat, 299-

308 membrane, electrical qualities of,

321-326 rat, 247-256 reinnervation, Synapse formation

and repression in, 299-308 Mutations, signal sequence, suppres-

sors of, 10-13 Myeloma, mouse, 363 Myosin, 96-98, 388 Myotonia, hereditary, 321

NAD, 179 NADH-cytochrome b5 reductase, 479-

491 Nafenopin (carcinogen), 422

Index / 619

Narcine. See Electric ray Nerve condition, pharmacologic block

of, 321-326 Nerve growth factor (NGF), 315, 338-

339 Nerve regeneration, 299-308 Neuraminidase, 601-605 Neuroblastoma cells, 211-234, 312-315,

317 Neuromuscular junction in developing

rat, 247-256 Neuromuscular Synapse, frog, 239-244 Neuronal cell differentiation, 311-317 Neurones, 339-343 Neurospora, 38-46 Nigericin, 271, 278, 280-284 Norepinephrine, 340-345, 578

Oligomeric protein assembly and trans­port, 27-34

Oligosaccharides, high-mannose, 445-455

Oncogenic RNA viruses, 367 Oocyte(s)

insulin action on, 163-167 maturation in Xenopus laevis, 157-

167 meiosis reimitation, calcium in, 139—

150 as model for growth regulation, 167 sea urchin, 171-181

Ornithine decarboxylase, 370, 593 Ouabain, 367

binding, 72-74, 79-84 -sensitive potassium ion uptake,

228-229 Pancreas, morphogenesis of, 413-426 Papaverine, 157 Paracentrotus lividus. See Sea urchin Peptides, nascent, translocation across

ER, 24-27 Permeability, membrane, in neuro­

blastoma cells, 224-227 Pharmacologic nerve block, 321-326 Phenothiazines, 548

Phenylhydrazine-induced anemia, 61, 64-67

Pheochromocytoma cells (PC 12), 308, 315-317

Phloretin, 331 Phlorizin, 331 Pholas, 140-142 Phosphatidyl ethanolamine, 116-117 Phosphatidylcholine, 116-117 Phosphatidylserine, 115-117 Phospholipase, 177-178 Phosphodiesterase, 148-149, 157 Phosphoglucomutase, 514 Photoreceptor cells, 352-353 Pig, kidney cell line LLC-PK,, 526-535,

543-549, 551-566 Placental lactogen, 390 Plasma membrane

glycoconjugate metabolism, 459-476 lateral diffusion, 525-535 steroid-growth hormone interaction

with, 157-167 Plasmalemmal glycoproteins, 413-426 Platymonas, 191 Pleurodeles, 147 Polarization of cultured thyroid cells,

403-411 Polyneuronal elimination, 247-256 Polyneuronal Innervation, 247-256 Polypeptide transmembrane transfer, 9 Potassium

ions, 278 and growth control in neuro­

blastoma cells, 214-216, 224-231

role in egg activation, 201-207 and salt transport in MDCK cells,

584-595 uptake, ouabain-sensitive, 228-

229 see a l s o Sodium

Prekeratin, 92-93, 96-98 Progesterone, 141-149, 157-167, 389-

390 Prolactin, 390, 393-400 Proline, 420

620 / Index

Prostaglandin(s), 578, 580-581, 590-594

E2, 314 F 2 a , 393

Protein A, 487 Protein(s)

anion transport, 59-69 band4„ 121-133 deficiency, erythrocyte membrane,

121-133 export in E. coli, 15-20 G/F, 160-161 intracellular postsynaptic, 239-244 localization, 4-10, 20 membrane, assembly in, 23-34 mitochondrial

membrane, biogenesis of, 37-46 transport, 49-58

NADH-cytochrome b5 reductase, 479-491

oligomeric, assembly and transport, 27-34

-phospholipid interaction, 113-117 transport, signal hypothesis of, 3-13 see a l s o Glycoproteins

Proteoglycans, 430, 435 Puromycin, 269-270, 278 Pyridoxal-P, 331 Pyrophosphatase, 489 Rabbit

BB isozyme, 387-388 mammary cell hormone responsive-

ness, 389-400 Rana pipiens, 143, 148 Rat

extensor digitorum longus muscle, 299-308

fibroblasts, cultured, 497-501 hepatic cells, 459-476 liver, 479-491 soleus muscle, 247-256

Ray, electric (Torpedo, Narcine), 240-244, 261, 264-265, 274, 287-294

RCA, 418 Receptor(s)

acetylcholine, 338 and calcium, 252-253, 256 in cultured muscle, 259-264, 278-

284 and growth regulation, 378, 383,

388 immunologic study of, 239-244 membrane localization of, 287-

294 EGF, monoclonal antibodies vs,

362-366 lambda, 3-13 lectin, 416-418 -mediated endocytosis and iron

metabolism, 495-518 mitochondrial, 39

Red cell(s) deformability, 124, 128-130 and iron metabolism, 496

Regeneration, nerve, 299-308 Rhabdovirus, 609 Salt transport, monovalent, in MDCK

cells, 584-595; see a l s o Calcium, Potassium, Sodium

Sea urchin, 140-142, 144, 171-181 embryo gastrulation, 420 spermatozoa, 185-186

Secretory component, 390, 392 Selenium, 578 Self-assembly mechanisms, 15-16 Serum

fetal calf, 212-215 Stimulation of neuroblastoma cells,

212-216 Sialoglycoconjugates, 418 Signal hypothesis, 3-13, 15-20 Signal sequence mutations, suppressors

of, 10-13 Siphonophores, sperm-egg attraction,

185-195 SITS, 331 Sleep and brain metabolism, 329-334 Sodium ions, 278

and growth control in MDCK cells, 584-595

Index / 621

in neuroblastoma cells, 214-216, 224-231

and hexose transport, 551-566 potassium pump activity in neuro­

blastoma cells, 227-231 role in egg activation, 197-207 see a l s o ATPase, (Na + ,K + ) ; Salt

transport Spectrin, 61, 65, 107-117, 122, 127 Speract, 185 Sperm acrosome reaction, 139 Spermatozoa

attraction to egg in siphonophores, 185-195

binding in echinoderms, 192-195 sea urchin, 185-186 swimming Stimulation, 185-186

Spherocytosis, hereditary, 121 Sphingomyelin, 116-117 Spisula, 140-142 Staphylococcus aureus rosettes, 64-65 Starfish, 140-145, 149, 171 Steroid(s)

adrenal, 542 -growth hormone interaction with

plasma membrane, 157-167 see a l s o Hormone(s), specific

Steroids Succinyl, 107 Synapse(s)

formation, 337-339 formation and repression in muscle

reinnervavation, 299-308 frog neuromuscular, 239-244 reorganization, 247-256

Taurine in CNS development, 349-354 Teratocarcinoma cells, mouse, 501 12-0-Tetradecanoylphorbol-13-acetate

(TPA), 555-558 Tetrodotoxin (TTX), 322-326 Thalassemia, and iron overload, 495-

518 Theophylline, 157, 555-557 Thyroglobulin, 403, 407 Thyroid, 403-411

Thyrotropin, 403, 405, 407 Tight junction, 525-535, 553, 575 Toad

bladder, 537-542 cell line, TB6c, 538-542 cell line TBM, 538-542 kidney, 537-542 kidney cell line A6, 526-535

Torpedo. See Electric ray Toxin(s)

o>Bungarotoxin, 240, 261-264, 277-281, 289, 383

cholera, 314 tetrodotoxin (TTX), 322-326 see a l s o Endotoxins

Transferrin, 390, 392-400, 578, 580-581 Translocation of nascent peptides

across ER, 24-27 Transmitter release in rat muscle, 255 Trifluoperazine, 150, 548 Trigger hypothesis, 15-20 Triiodothyronine, 578, 581, 592 Triiodotyronine, 393 Trypsin-insensitive domain (TID), 289 Tubularia, 189 Tumor Promoters, 555, 566. See a l s o

Carcinogenesis; Virus(es), oncogenic

Tumorigenicity of various cell lines, 572 Tunicamycin, 274-275, 278-279, 418-

419, 436 Urechis, 140-142

Vasopressin, 543-549, 578 Vectorial translation, 50 Verapamil, 174 Vesicular Stomatitis virus, 59, 600-609 Villin, 90-93, 98 Vimentin, 241 Vinblastine, 150, 321 Vincristine, 321 Virus(es)

envelope proteins, 480 enveloped, 102 enveloped, entry into epithelial cell

622 / Index

line, 599-609 fowl plague, 600-609 influenza, 600-603 Moloney sarcoma, 575 Oligosaccharides, biosynthesis of,

445-455 oncogenic RNA, 367 rhabdovirus, 609 Semliki Forest, 59, 454, 599-609 Sendai, 599 Sindbis, 59, 445-455

SV40, 572 Toga, 454 vesicular Stomatitis, 59, 600-609

von Gierke disease, 330-331 X537A, 271, 278, 280-284 Xenopus laevis, 143, 148, 157-167 Yeast, 46

mitochondria, 49-58 petite, 51-56