Protein Secretion: Methods and Protocols

by Anastassios Economou (Editor)

Overview

Over the past thirty years, many elegant genetic and biochemical approaches have been combined in order to advance the study of protein secretion and the necessary navigation through cell membranes, yet, despite this progress, less than two hundred membrane protein structures are known, nowhere near the complete inventory that the discovered protein export systems suggest. In Protein Secretion: Methods and Prools, leading experts in the field provide robust, well-established prools to elucidate the multiplicity of tools that have been developed to study protein sorting, membrane targeting, transmembrane crossing, and secretion across multiple membranes. With examples involving both prokaryotic and eukaryotic organisms, the volume covers subjects ranging from bioinformatics and proteomics to fundamental enzymology and genetics to cell biology, structural analyses, and biophysics. Written in the highly successful Methods in Molecular Biology™ series format, chapters contain introductions to their respective topics, lists of the key materials and reagents, step-by-step, readily reproducible prools, and detailed notes on troubleshooting and avoiding known pitfalls.

Comprehensive and dependable, Protein Secretion: Methods and Prools focuses on well-characterized paradigms so that scientists studying a vast array of subjects from biochemistry and genetics to biotechnology and biopharmaceuticals can benefit and expand upon their vital research.

Product Details

ISBN-13:	9781603271677
Publisher:	Springer-Verlag New York, LLC
Publication date:	04/26/2010
Series:	Methods in Molecular Biology , #619
Edition description:	2010
Pages:	426
Product dimensions:	7.20(w) x 9.90(h) x 1.40(d)

Table of Contents

Preface v

Contributors xi

1 The Extraordinary Diversity of Bacterial Protein Secretion Mechanisms I. Barry Holland 1

2 In Vitro and In Vivo Approaches to Studying the Bacterial Signal Peptide Processing Peng Wang Ross E. Dalbey 21

3 Membrane Insertion of Small Proteins Andreas Kuhn Natalie Stiegler Anne-Kathrin Schubert 39

4 Membrane Protein Insertion in E. coli Jijun Yuan Ross E. Dalbey Andreas Kuhn 63

5 Study of Polytopic Membrane Protein Topological Organization as a Function of Membrane Lipid Composition Mikhail Bogdanov Philip N. Heacock William Dowhan 79

6 In Vivo Analysis of Protein Translocation to the Escherichia coli Periplasm Dominique Belin 103

7 Sorting of Bacterial Lipoproteins to the Outer Membrane by the Lol System Shin-ichiro Narita Hajime Tokuda 117

8 Purification and Functional Reconstitution of the Bacterial Protein Translocation Pore, the SecYEG Complex Ilja Kusters Geert van den Bogaart Janny de Wit Viktor Krasnikov Bert Poolman Arnold Driessen 131

9 Reconstitution of the SecY Translocon in Nanodiscs Kush Dalal Franck Duong 145

10 In Vitro Assays to Analyze Translocation of the Model Secretory Preprotein Alkaline Phosphatase Giorgos Gouridis Spyridoula Karamanou Marina Koukaki Anastassios Economou 157

11 Characterization of Interactions Between Proteins Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy Jennine M. Crane Angela A. Lilly Linda L. Randall 173

12 Analysis of Tat Targeting Function and Twin-Arginine Signal Peptide Activity in Escherichia coli Tracy Palmer Ben C. Berks Frank Sargent 191

13 Site-Specific Cross-Linking of In Vitro Synthesized E. coli Preproteins for Investigating Transmembrane Translocation Pathways Sascha Panahandeh Matthias Müller 217

14 Tracking the Secretion of Fluorescently Labeled Type III Effectors from Single Bacteria in Real Time Nandi Simpson Laurent Audry Jost Enninga 241

15 Comparative Analysis of Cytoplasmic Membrane Proteomes of Escherichia coli Using 2D Blue Native/SDS-PAGE Susan Schlegel Mirjam Klepsch David Wickström Samuel Wagner Jan-Willem de Gier 257

16 Using Hidden Markov Models to Discover New Protein Transport Machines Vladimir A. Likic Pavel Dolezal Nermin Celik Michael Dagley Trevor Lithgow 271

17 Bioinformatics Predictions of Localization and Targeting Shruti Rastogi Burkhard Rost 285

18 The Chloroplast Protein Import Machinery: A Review Penelope Strittmatter Jürgen Soll Bettina Bölter 307

19 Measurement of the Energetics of Protein Transport Across the Chloroplast Thylakoid Membrane Steven M. Theg 323

20 In Vitro Dissection of Protein Translocation into the Mammalian Endoplasmic Reticulum Ajay Sharma Malaiyalam Mariappan Suhila Appathurai Ramanujan S. Hegde 339

21 In Vitro Reconstitution of the Selection, Ubiquitination, and Membrane Extraction of a Polytopic ERAD Substrate Kunio Nakatsukasa Jeffrey L. Brodsky 365

22 Studying the ArfGAP-Dependent Conformational Changes in SNAREs Fernanda Rodriguez Anne Spang 377

23 Studying Endoplasmic Reticulum Function In Vitro Using siRNA Cornelia M. Wilson Stephen High 389

24 High-Quality Immunofluorescence of Cultured Cells Dibyendu Bhattacharyya Adam T. Hammond Benjamin S. Glick 403

25 Trapping Oxidative Folding Intermediates During Translocation to the Intermembrane Space of Mitochondria: In Vivo and In Vitro Studies Dionisia P. Sideris Kostas Tokatlidis 411

26 Native Techniques for Analysis of Mitochondrial Protein Import F.-Nora Vögtle Oliver Schmidt Agnieszka Chacinska Nikolaus Pfanner Chris Meisinger 425

Subject Index 437