Laboratory Methods in Vesicular and Vectorial Transport - 1st Edition - ISBN: 9780126837551, 9780323146791

Laboratory Methods in Vesicular and Vectorial Transport

1st Edition

Editors: Alan Tartakoff
eBook ISBN: 9780323146791
Imprint: Academic Press
Published Date: 28th October 1991
Page Count: 536
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Laboratory Methods in Vesicular and Vectorial Transport describes the procedures used to study the mechanisms of vesicular transport along the secretory and endocytic paths, including electron microscopy, autoradiography, and methods associated with cyto- and immunocytochemistry, genetics, and biochemistry. It investigates vectorial transport to the cisternal space of the rough endoplasmic reticulum (ER), as well as protein translocation across the ER, strategies for gaining access to the cytoplasm, cell-free analysis of vesicle fusion, the structure of glycoproteins, and the use of cell systems for analysis of vesicular traffic. Organized into seven parts encompassing 20 chapters, this volume begins with an overview of protein topology in the ER and the use of cross-linking methods to probe the molecular environment of translocating polypeptide chains. It then discusses the reconstitution of secretory protein translocation from detergent-solubilized rough microsomes; the use of anti-idiotype antibodies to characterize protein-protein interactions; the use of perforated cells to elucidate intracellular membrane transport; delivery of macromolecules into cells expressing a viral membrane fusion protein; and digitonin permeabilization procedures for studying endosome acidification and function. The reader is also introduced to reconstitution of intracellular vesicle fusion in a cell-free system after receptor-mediated endocytosis; immunoisolation using magnetic solid supports; endosome and lysosome purification by free-flow electrophoresis; remodeling of glycoprotein oligosaccharides after endocytosis; and replica plating of animal cells. This book will interest students, researchers, geneticists, biochemists, and cell biologists.

Table of Contents

Contents of Methods in Cell Biology Volumes 31, 32, and 34



Part I. Vectorial Transport to the Cisternal Space of the Rough Endoplasmic Reticulum

Analysis of Protein Topology in the Endoplasmic Reticulum

I. Introduction

II. Insertion of Wild-Type and Mutant Proteins into the ER

III. Probing the Transmembrane Topology

IV. Discussion


Transcription of Full-Length and Truncated mRNA Transcripts to Study Protein Translocation across the Endoplasmic Reticulum

I. Introduction

II. In Vitro Transcription of mRNA

III. Translation of mRNA Transcripts

IV. Ribonucleotide-Dependent Translocation Systems


Probing the Molecular Environment of Translocating Polypeptide Chains by Cross-Linking

I. Introduction

II. The Cross-Linking Methods—General Considerations

III. Cross-Linking Protocols

IV. Glossary


Reconstitution of Secretory Protein Translocation from Detergent-Solubilized Rough Microsomes

I. Introduction

II. Preparation of Membranes

III. Reconstitution of Translocation-Competent Vesicles

IV. Structural Characterization of Reconstituted Membranes

V. Analysis of Activity: Reconstituted Membranes

VI. Detergent Requirements for Reconstitution

VII. Discussion/Conclusions


Part II. Gaining Access to the Cytoplasm

Perforated Cells for Studying Intracellular Membrane Transport

I. Introduction

II. Generation and Characterization of Perforated Cells

III. Reconstitution of Intracellular Membrane Transport

IV. Discussion


Delivery of Macromolecules into Cells Expressing a Viral Membrane Fusion Protein

I. Introduction

II. Development of Cell Lines that Express the Influenza HA

III. Red Blood Cell-Mediated Delivery

IV. Liposome-Mediated Delivery

V. Advantages and Disadvantages of the Method

VI. Protocols


Digitonin Permeabilization Procedures for the Study of Endosome Acidification and Function

I. Introduction

II. Preparation of Mannose-BSA for Endosome Acidification Studies

III. Permeabilization of Cells

IV. Endosome Acidification

V. Summary and Outlook


Part III. Cell-Free Analysis of Vesicle Fusion

Reconstitution of Intracellular Vesicle Fusion in a Cell-Free System after Receptor-Mediated Endocytosis

I. Introduction

II. Generation of Probes

III. Vesicle Preparation

IV. General Requirements for Endosome-Endosome Fusion

V. Cytosol and Membrane-Associated Factors

VI. Conclusions


Immunoisolation Using Magnetic Solid Supports: Subcellular Fractionation for Cell-Free Functional Studies

I. Introduction

II. Immunoisolation

III. Immunoisolation of the ComPartments of the Endocytic Pathway

IV. Immunoisolated Endosomal Fractions in Cell-Free Assays of Vesicle Fusion

V. Perspectives


Endosome and Lysosome Purification by Free-Flow Electrophoresis

I. Introduction

II. Free-Flow Electrophoresis

III. Sample Preparation

IV. Subfractionation of Endosomes by FFE

V. Conclusions and Prospects


Part IV. Electron Microscopic Immunocytochemistry

Immunoperoxidase Methods for the Localization of Antigens in Cultured Cells and Tissue Sections by Electron Microscopy

I. Introduction

II. Localization of Antigens within Cultured Cells

III. Special Considerations for the Localization of Antigens within Cells of Tissue Sections

IV. Summary


Postembedding Labeling on Lowicryl K4M Tissue Sections: Detection and Modification of Cellular Components

I. Introduction

II. Some Physicochemical Characteristics of Lowicryl K4M

III. Low-Temperature Embedding in Lowicryl K4M

IV. Sectioning and Section Storage

V. Protocols for Labeling on Sections

VI. Enzymatic and Chemical Modifications on Lowicryl K4M Sections

VII. Prevention of Artifacts


Postembedding Detection of Acidic ComPartments

I. Introduction

II. Materials and Methods

III. Results and Discussion

IV. Conclusions


Part V. Analysis of Glycoprotein Structure

Remodeling of Glycoprotein Oligosaccharides after Endocytosis: A Measure of Transport into ComPartments of the Secretory Apparatus

I. Introduction

II. Transport of Glycoproteins to Sialyltransferase-Containing ComPartments

III. Transport of Glycoproteins to Golgi ?-Mannosidase I-Containing ComPartments

IV. Conclusion


Enzymatic Approaches for Studying the Structure, Synthesis, and Processing of Glycoproteins

I. Introduction

II. Oligosaccharide-Cleaving Enzymes

III. Related Matters


Separation and Analysis of Glycoprotein Oligosaccharides

I. Introduction

II. Diversity of Sugar Chains in Animal Cell Glycoproteins

III. Evidence Indicating that a Protein Is Glycosylated

IV. Radiolabeling of Sugar Chains

V. Enzymatic and Chemical Methods for Releasing Sugar Chains from Proteins

VI. Separation of Sugar Chains by Serial Lectin Affinity Chromatography

VII. Analysis of Metabolically Radiolabeled Sugar Chains

VIII. Conclusions


Part VI. Cell Systems for Analysis of Vesicular Traffic

Using Temperature-Sensitive Mutants of VSV to Study Membrane Protein Biogenesis

I. Introduction

II. Synchronizing the Transport of the ts045 G Protein

III. Prospects for Future Uses of Temperature-Sensitive Mutants

IV. Technical Procedures


Analysis of the Synthesis, Intracellular Sorting, and Function of Glycoproteins Using a Mammalian Cell Mutant with Reversible Glycosylation Defects

I. Introduction

II. Synthesis and Function of Protein-Linked Oligosaccharides

III. CHO Mutants with Defects in Oligosaccharide Biosynthesis

IV. Reversibility of the Glycosylation Defects in IdlD Cells

V. Glycoprotein Synthesis, Sorting, and Function Studied Using IdlD Cells

VI. Care and Feeding of IdlD Cells


Replica Plating of Animal Cells

I. Introduction

II. Replica Plating of Animal Cells

III. Preparation of Disks, Beads, and Master Plates

IV. Isolation of Mutants by Indirect Screenings

V. Conclusions


Part VII. Production and Use of Antiidiotypic Antibodies

The Use of Antiidiotype Antibodies for the Characterization of Protein-Protein Interactions

I. Introduction

II. Identification of the KDEL Receptor

III. Budding of Alphaviruses

IV. Conclusion




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© Academic Press 1991
Academic Press
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About the Editor

Alan Tartakoff

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