Subcellular Biochemistry Volume 19 Endocytic Components: Identification and Characterization

SUBCELLULAR BIOCHEMISTRY

SERIES EDITOR J. R. HARRIS, Institute of Zoology, University of Mainz, Mainz, Germany

ASSISTANT EDITORS H. J. HILDERSON, University of Antwerp, Antwerp, Belgium D. A. WALL, SmithKline Beecham Pharmaceuticals,

King of Prussia, Pennsylvania, U.S.A.

Recent Volumes in This Series:

Volumes 5-11 Edited by Donald B. Roodyn

Volume 12 Immunological Aspects Edited by J. R. Harris

Volume 13 Fluorescence Studies on Biological Membranes Edited by H. J. Hilderson

Volume 14 Artificial and Reconstituted Membrane Systems Edited by J. R. Harris and A.-H. Etemadi

Volume 15 Virally Infected Cells Edited by J. R. Harris

Volume 16 Intracellular Transfer of Lipid Molecules Edited by H. J. Hilderson

Volume 17 Plant Genetic Engineering Edited by B. B. Biswas and J. R. Harris

Volume 18 Intracellular Parasites Edited by J. L. Avila and J. R. Harris

Volume 19 Endocytic Components: Identification and Characterization Edited by J. J. M. Bergeron and J. R. Harris

A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher.

Subcellular Biochemistry Volume 19 Endocytic Components: Identification and Characterization Edited by

J. J. M. Bergeron Department of Anatomy McGill University Montreal, Canada

and J. R. Harris Institute of Zoology University of Mainz Mainz, Germany

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

The Library of Congress cataloged the first volume of this title as folIows: Sub-cellular biochemistry.

London, New York, Plenum Press. v. illus. 23 cm. quarterly.

Began with Sept. 1971 issue. Cf. New serial titles. 1. Cytochemistry - Periodicals. 2. Cell organelles - Periodicals.

QH611.S84 574.8'76

ISBN 978-1-4613-6319-4 ISBN 978-1-4615-3026-8 (eBook) DOI 10.1007/978-1-4615-3026-8 This series is a continuation of the journal Sub-Cellular Biochemistry, Volumes 1 to 4 of which were published quarterly from 1972 to 1975

© 1993 Springer Science+Business Media New York Originally published by Plenum Press, New York in 1993

All rights reserved

73-643479

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Softcover reprint of the hardcover 1st edition 1993

INTERNATIONAL ADVISORY EDITORIAL BOARD

J. L. AVILA, Instituto de Biomedicina, Caracas, Venezuela

J. J. M. BERGERON, McGill University, Montreal, Canada

B. B. BISWAS, Bose Institute, Calcutta, India

N. BORGESE, CNR Center for Pharmacological Study, Milan, Italy

M. J. COSTELLO, University of North Carolina, Chapel Hill, North Carolina, USA

A.-H. ETEMADI, University of Paris VI, Paris, France

W. H. EVANS, National Institute for Medical Research, London, England

P. L. J0RGENSEN, University of Aarhus, Aarhus, Denmark

J. B. LLOYD, University of Keele, Keele, England

J. A. LUCY, Royal Free Hospital School of Medicine, London, England

A. H. MADDY, University of Edinburgh, Edinburgh, Scotland

J.D. MORRE, Purdue University, West Lafayette, Indiana, USA

P. QUINN, King's College London, London, England

G. RALSTON, The University of Sydney, Sydney, Australia

S. ROTTEM, The Hebrew University, Jerusalem, Israel

M. R. J. SALTON, New York University Medical Center, New York, New York, USA G. SCHATTEN, University of Wisconsin-Madison, Madison, Wisconsin, USA

F. WUNDERLICH, University of Dusseldorf, Dusseldorf, Germany

I. B. ZBARSKY, Russian Academy of Sciences, Moscow, Russia

Contributors

J. J. M. Bergeron Department of Anatomy, McGill University, Montreal, Canada H3A 2B2

J. S. Blum Department of Cell Biology and Physiology, Washington Uni-versity School of Medicine, St. Louis, Missouri 63110. Current ad-dress: Virginia Mason Research Center, Seattle, Washington 98101

J. W. Burgess Department of Anatomy, McGill University, Montreal, Can-ada H3A 2B2

Pierre J. Courtoy Cell Biology Unit, International Institute of Cellular and Molecular Pathology and University of Louvain Medical School, 1200 Brussels, Belgium

R. Diaz Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Carlos Enrich Departmento de Biologia Celular, Facultad Medicina, Uni-versitat de Barcelona, Spain

W. Howard Evans National Institute for Medical Research, Mill Hill, Lon-don NW7 lAA, United Kingdom

Michel Jadot Laboratoire de Chimie Physiologique, Facultes Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium

M. N. Khan Department of Anatomy, McGill University, Montreal, Canada H3A 2B2. Current address: Phoenix International Sciences, Montreal, Canada H4R 2N6

W. H. Lai Department of Anatomy, McGill University, Montreal, Canada H3A 2B2

Frederick R. Maxfield Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

L. S. Mayorga Facultad de Cincias Medican, Instituto de Histologia y Em-briologia, 550 Mendoze, Argentina

vii

viii Contributors

Sandra Misquith Laboratoire de Chimie Physiologique, Facultes Univer-sitaires Notre-Dame de la Paix, B-5000 Namur, Belgium

B. I. Posner Department of Medicine, McGill University, Montreal, Cana-da H3A 2B2

Nita H. Salzman Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, and Depart­ment of Pharmacology, New York University School of Medicine, New York, New York 10016

Sandra L. Schmid Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

P. D. Stahl Department of Cell Biology and Physiology, Washington Uni-versity School of Medicine, St. Louis, Missouri 63110

Robert Wattiaux Laboratoire de Chimie Physiologique, Facultes Univer-sitaires Notre-Dame de la Paix, B-5000 Namur, Belgium

Simone Wattiaux-de Coninck Laboratoire de Chimie Physiologique, Fac-ultes Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium

Paul H. Weigel Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0647

Preface

The rapid advance of knowledge relating to the endosome, its cellular localiza­tion, separation, and functions, has led to the appropriate inclusion of this excit­ing topic within the Subcellular Biochemistry series. Furthermore, the continuity of emphasis on "membrane-related" topics within the series is thereby main­tained. A list of internationally respected authors has been compiled, with the aim of covering within a single book the principal areas currently being explored within the field of endosome research.

The book commences with a chapter by S. L. Schmid, who describes in detail the use of free flow electrophoresis for the physical separation and bio­chemical definition of the endosomal compartment. P. J. Courtoy then deals specifically with the endosomal compartment in rat hepatocytes; he covers a range of analytical approaches in an attempt to derive an overall structural model of the rat liver endosome. This is followed by a chapter by J. S. Blum and colleagues in which they deal with proteolysis in endosomes and discuss its significance for vesicle fusion and vesicle-mediated transport during endocytosis in reconstituted systems. The application of a range of different quantitative fluorescence techniques for the characterization of endocytosis is presented by N. H. Salzman and F. R. Maxfield, who thoroughly survey this important technical approach. This is followed by a chapter by P. H. Weigel on endocytosis and the function of the hepatic asialoglycoprotein (galactosyl/N-acetylgalac­tosaminyl) receptor. The endocytic components of liver nonparenchymal cells are characterized in detail by R. Wattiaux and colleagues, who present a compre­hensive survey of the endocytic behavior of this population of cells. Comple­menting this chapter is one by C. Enrich and W. H. Evans in which the endocytic compartment of normal and regenerating liver is discussed. In the final chapter, M. N. Khan and colleagues discuss the potential role of endosomes in trans­membrane signaling. Having defined the organelles involved in receptor-medi-

ix

X Preface

ated endocytosis, these authors are concerned primarily with the functions of endosomes in cellular signaling.

It is hoped that the broad coverage presented by the material included in the eight chapters of this book will enable it to be of interest and use to cell biologists and biochemists concerned with this rapidly advancing field, as well as other scientists wishing to keep abreast of progress in this area of membrane biochem­istry. In addition, the book should be of use to honors degree and graduate students in cell biology and biochemistry.

Montreal, Canada, and Mainz, Germany

J. J. M. Bergeron J. R. Harris

Contents

Chapter 1 Toward a Biochemical Definition of the Endosomal Compartment: Studies Using Free Flow Electrophoresis

Sandra L. Schmid

1. Overview .............................................. . 2. Toward a Biochemical Definition of the Organelles of the

Endocytic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.1. Organelles of the Biosynthetic Pathway Are Reasonably

Well Defined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.2. Organelles of the Endocytic Pathway Are Poorly Defined . . 3 2.3. Operational Definitions for the Organelles along the

Endocytic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. Large-Scale Purification of Endosomes from Tissue Culture Cells 6

3.1. Purification of Endosome Subpopulations by Free Flow Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.2. A Rapid Analytical Method for Analysis of Protein Transport through Endosomal Subpopulations . . . . . . . . . . . . 15

4. Advantages and Disadvantages of Free Flow Electrophoresis . . . . . 15 5. Comparisons with Other Methods for Endosome Purification . . . . . 18 6. What Have We Learned? A Model for Membrane Transport along

the Endocytic Pathway in CHO Cells . . . . . . . . . . . . . . . . . . . . . . . . 20 7. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

7 .I. Other Approaches for the Use of FFE . . . . . . . . . . . . . . . . . . 23 7.2. The Search for an Endosome-Specific Marker . . . . . . . . . . . 24

8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

xi

xii Contents

Chapter 2 Analytical Subcellular Fractionation of Endosomal Compartments in Rat Hepatocytes

Pierre J. Courtoy

1. Definition of the Endosomal Compartments . . . . . . . . . . . . . . . . . . . 29 1.1. Heterogeneity of Endosomal Compartments in Rat

Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1.2. Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 1.3. Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2. Analytical Subcellular Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.1. Methodological Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.2. Differential Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 2.3. Isopycnic Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.4. In Vivo Density Modifications . . . . . . . . . . . . . . . . . . . . . . . . 40 2.5. In Vitro Density Modifications . . . . . . . . . . . . . . . . . . . . . . . . 41 2.6. Use of Surface Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3. Composition and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.1. Intrinsic Membrane Polypeptides . . . . . . . . . . . . . . . . . . . . . . 45 3.2. Membrane Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3.3. Soluble Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 3.4. Posttrans1ational Modifications . . . . . . . . . . . . . . . . . . . . . . . . 49 3.5. Sorting, Segregation, and Addressing . . . . . . . . . . . . . . . . . . 52

4. A Structural Model of Rat Hepatocyte Endosomes . . . . . . . . . . . . . 53 4.1. Primary Basolateral Endosomes . . . . . . . . . . . . . . . . . . . . . . . 55 4.2. Secondary (Tubulospherical) Endosomes . . . . . . . . . . . . . . . . 55 4.3. Tertiary (Multivesicular, Late) Endosomes . . . . . . . . . . . . . . 56 4.4. Recycling Basolateral Endosomes . . . . . . . . . . . . . . . . . . . . . 57 4.5. Transcytotic Endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.6. Apical Endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Chapter 3 Reconstitution of Endosomal Transport and Proteolysis

J. S. Blum, R. Diaz, L. S. Mayorga, and P. D. Stahl

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2. Proteolysis in Endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

2.1. Characteristics of Endosomal Proteolysis . . . . . . . . . . . . . . . . 71 2.2. Identification of Specific Proteases Found in Endosomes . . . 73 2.3. Biological Importance of Endosomal Proteases . . . . . . . . . . . 75

Contents xiii

3. Reconstitution of Vesicle Fusion During Endocytosis . . . . . . . . . . . 77 3.1. Endosome-Endosome Fusion . . . . . . . . . . . . . . . . . . . . . . . . . 79 3.2. Endosome-Plasma Membrane Fusion . . . . . . . . . . . . . . . . . . 83 3.3. Proteolysis . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 85

4. Regulation of Vesicle-Mediated Transport During Endocytosis . . . . 86 4.1. NEM-Sensitive Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.2. Role of GTP-Binding Proteins in the Fusion Process . . . . . . 87

5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Chapter 4 Quantitative Fluorescence Techniques for the Characterization of Endocytosis in Intact Cells

Nita H. Salzman and Frederick R. Maxfield

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 2. Fluorescent Probes of Fluid-Phase Pinocytosis . . . . . . . . . . . . . . . . . 99 3. Probes of Receptor-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . 99 4. Fluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 5. Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 6. Microscope Spectrofluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 7. pH Measurements of Endocytic Compartments . . . . . . . . . . . . . . . . 103 8. Fluorescence Studies of Ligand-Receptor Sorting . . . . . . . . . . . . . . 106 9. Endocytosis of Fluorescently Labeled Lipids . . . . . . . . . . . . . . . . . . 115

10. A Model for Efficient Sorting of Ligands and Receptors . . . . . . . . 117 11. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 12. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Chapter 5 Endocytosis and Function of the Hepatic Asialoglycoprotein Receptor

Paul H. Weigel

1. The Hepatocyte Asialoglycoprotein (Galactosyl/N-Acetylgalactosaminyl) Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 1.1. General Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 1.2. Specificity of the Gal Receptor . . . . . . . . . . . . . . . . . . . . . . . 126 1.3. Polypeptide Composition of the Gal Receptor . . . . . . . . . . . . 126 1.4. Proposed Structure of the Gal Receptor . . . . . . . . . . . . . . . . . 127

2. Multiple Pathways for Receptor-Mediated Endocytosis . . . . . . . . . . 129 2.1. The Hepatic Gal-Receptor System Functions via Two

Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

xiv Contents

2.2. Other Receptor Systems Function via Two Pathways 134 3. Constitutive Receptor Recycling and the Gal-Receptor

Inactivation/Reactivation Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 3.1. Discovery of a Receptor Inactivation/Reactivation Cycle . . . 135 3.2. Uncoupling of State 2 Gal-Receptor Inactivation and

Receptor Redistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 3.3. The Role of Gal-Receptor Phosphorylation . . . . . . . . . . . . . . 140

4. Possible Functions of the Endocytic Membrane-Bound Carbohydrate Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 4.1. The Mannose-6-Phosphate (Man-6-P) Receptor(s) . . . . . . . . 141 4.2. The Reticuloendothelial Mannosyl (Man) Receptor . . . . . . . 141 4.3. The Macrophage Man/GlcNAc Receptor . . . . . . . . . . . . . . . . 142 4.4. The Kupffer Cell Fucosyl (Fuc) Receptor . . . . . . . . . . . . . . . 143 4.5. The Macrophage Gal Receptor . . . . . . . . . . . . . . . . . . . . . . . . 143 4.6. The Liver Endothelial Cell Hyaluronan/Glycosaminoglycan

Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 4. 7. The Glucosyl-Advanced Glycation End Products (AGE)

Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 5. Function of the Hepatic Asialoglycoprotein Receptor . . . . . . . . . . . 145

5.1. Previously Proposed Functions of the Hepatic Parenchymal Gal/GalNAc Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

5.2. The Galactosyl Homeostasis Hypothesis . . . . . . . . . . . . . . . . 147 6. References.................... . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Chapter 6 Characterization of Endocytic Components of Liver Nonparenchymal Cells

Robert Wattiaux, Michel Jadot, Sandra Misquith, and Simone Wattiaux­de Coninck

1. Introduction....................... . . . . . . . . . . . . . . . . . . . . . . 163 1.1. Liver Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 1.2. Receptor-Mediated Endocytosis by Liver Cells . . . . . . . . . . . 164

2. How to Study Endocytic Components in Liver Nonparenchymal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

3. Meth0(lological Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 3.1. Choice of the Endocytosed Molecule . . . . . . . . . . . . . . . . . . . 166 3.2. Distribution of Nonparenchymal Cell Organelles . . . . . . . . . 166

Contents XV

3.3. Distinction between Prelysosomal and Lysosomal Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

3 .4. Intracellular Degradation of the Endocytosed Compound . . . 171 4. Intracellular Journey of a Protein Endocytosed by Liver Sinusoidal

Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 4.1. Differential Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 4.2. lsopycnic Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 4.3. Use of Density Perturbants . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 4.4. Effect of Glycyl-L-Phenylalanine-2-Naphthylamide (Gly-L-

Phe-2-NNap) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 4.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

5. Intracellular Fate of tBSA Endocytosed by the Liver . . . . . . . . . . . 179 5.1. ML and P Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 5.2. Fractions Isolated by Gradient Centrifugation . . . . . . . . . . . . 180 5.3. Purified Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 5.4. In Vitro Degradation of tBSA . . . . . . . . . . . . . . . . . . . . . . . . 183 5.5. Identification of Proteolytic Activities . . . . . . . . . . . . . . . . . . 185

6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Chapter 7 The Endocytic Compartments of Normal and Regenerating Liver

Carlos Enrich and W. Howard Evans

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 2. Morphological Features of the Hepatic Endocytic Compartment . . 197 3. Biochemical Properties of Hepatic Endosomes . . . . . . . . . . . . . . . . 199

3.1. Proteins of the Liver Plasma Membrane and Endosomes: Establishing a Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

3.2. Distribution in Liver Membranes of Trimeric and Low-Molecular-Weight G Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 208

3. 3. Targeting to Plasma Membrane Domains . . . . . . . . . . . . . . . . 210 3.4. Polydispersity of Liver Endosomal Fractions . . . . . . . . . . . . . 211

4. Association of Endosomes with Cytoskeletal Components, Calmodulin, and Extracellular Matrix . . . . . . . . . . . . . . . . . . . . . . . . 213

5. Immunological Approaches Toward Identifying Endosome-Specific Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

6. Endocytosis in Regenerating Liver . . . . . . . . . . . . . . . . . . . . . . . . . . 214 7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

xvi Contents

Chapter 8 Potential Role of Endosomes in Transmembrane Signaling M. N. Khan, W. H. Lai, J. W. Burgess, B. I. Posner, and J. J. M. Bergeron

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 2. Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 3. Organelles Involved in Receptor-Mediated Endocytosis . . . . . . . . . 225

3 .1. Coated Pits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 3.2. Intracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 3.3. Endosomes: Terminology and Definition . . . . . . . . . . . . . . . . 227 3.4. Endosomes: Distinct or Temporally Continuous Component 229

4. Isolation and Purification of Endosomes . . . . . . . . . . . . . . . . . . . . . . 229 4.1. Biochemical Properties of Endosomes . . . . . . . . . . . . . . . . . . 233 4.2. Functions of Endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Index...................................................... 255