Laboratory Methods in Vesicular and Vectorial Transport

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.