Membrane Trafficking

As membrane trafficking research has expanded, a remarkable convergence of information has been gained by using genetic approaches in yeast cells with biochemical approaches in mammalian cells. This book reflects these advances by devoting one section of the book to yeast cells and the other to mammalian cells, with each section providing both classic and cutting-edge techniques to study macromolecular transport across the membranes. These protocols have been tested in laboratories all around world. As a volume of the highly successful Methods in Molecular Biology™ series, the chapters include step-by-step laboratory protocols, a list of necessary materials and reagents, and the greatly beneficial Notes section, which aids in troubleshooting and avoiding known pitfalls. Comprehensive and state-of-the-art, this is the ideal reference for both senior researchers experienced in the field and graduate students studying membrane trafficking for the first time.

Wide-ranging selection of methods for mammalian and yeast cells Methods for in vivo and in vitro assays High-throughput genome-wide approaches using yeast cells Microscopic and biochemical methods Contains protocols for the study of protein translocation across or into membranes of the endoplasmic reticulum, mitochondria, and nucleus Provides protocols for characterization of vesicle-mediated transport between endoplasmic reticulum, Golgi, lysosomes, and plasma membrane Methods represent both classic and cutting-edge techniques

Inhalt
Yeast Cells.- 1 Freight Management in the Cell: Current Aspects of Intracellular Membrane Trafficking.- High-Throughput Protein Extraction and Immunoblotting Analysis in Saccharomyces cerevisiae.- Genome-Wide Analysis of Membrane Transport Using Yeast Knockout Arrays.- A Cell-Free System for Reconstitution of Transport Between Prevacuolar Compartments and Vacuoles in Saccharomyces cerevisiae.- In vitro Analysis of the Mitochondrial Preprotein Import Machinery Using Recombinant Precursor Polypeptides.- In Vitro Import of Proteins Into Isolated Mitochondria.- Synthesis and Sorting of Mitochondrial Translation Products.- In Vivo Labeling and Analysis of Mitochondrial Translation Products in Budding and in Fission Yeasts.- Exploring Protein-Protein Interactions Involving Newly Synthesized Mitochondrial DNA-Encoded Proteins.- Purification of Yeast Membranes and Organelles by Sucrose Density Gradient Centrifugation.- Microscopic Analysis of Lipid Droplet Metabolism and Dynamics in Yeast.- Use of Bimolecular Fluorescence Complementation in Yeast Saccharomyces cerevisiae.- Mammalian Cells.- Using Quantitative Fluorescence Microscopy to Probe Organelle Assembly and Membrane Trafficking.- Measuring Secretory Membrane Traffic.- A Correlative Light and Electron Microscopy Method Based on Laser Micropatterning and Etching.- Approaches to Investigate the Role of Signaling in ER-to-Golgi Transport.- Recruitment of Coat Proteins to Peptidoliposomes.- SNARE-Mediated Fusion of LIposomes.- Use of Polarized PC12 Cells to Monitor Protein Localization in the Early Biosynthetic Pathway.- Tracking the Transport of E-Cadherin to and From the Plasma Membrane.- Analysis of Nucleocytoplasmic Shuttling of NF?B Proteins in Human Leukocytes.- Imaging pHluorin-Based Probes at Hippocampal Synapses.-Analysis of Receptor Tyrosine Kinase Internalization Using Flow Cytometry.- Measurement of Receptor Endocytosis and Recycling.- Styryl Dye-Based Synaptic Vesicle Recycling Assay in Cultured Cerebellar Granule Neurons.- Use of Quantitative Immunofluorescence Microscopy to Study Intracellular Trafficking: Studies of the GLUT4 Glucose Transporter.- Dissecting GLUT4 Traffic Components in L6 Myocytes by Fluorescence-Based, Single-Cell Assays.- Functional Genetic Analysis of the Mammalian Mitochondrial DNA Encoded Peptides.