H W Davidson

Transport of protein between endoplasmic reticulum and Golgi compartments in semiintact cells

Publisher Summary This chapter discusses the transport of protein between endoplasmic reticulum and golgi compartments in semiintact cells. Preparation of cells that actively transport VSV G protein are most thoroughly characterized using CHO cells and CHO cytosol. However, there is considerable variability in transport using different cell lines or conditions. Where indicated in the procedures, measurement of transport using different marker proteins, or the use of different cell lines or cytosol preparations, may need to be optimized to obtain maximal efficiencies of transport. Extensive fragmentation and lysis of the ER (particularly in the case of strongly adherent cells) generally result in reduced transport. In particular, perforation conditions can lead to the release of soluble marker proteins from the ER during preparation of semiintact cells. In contrast, poor perforation leads to a transport that is efficient, but cytosol independent. In the latter case, cytosol dependence can be enhanced by gently homogenizing the semiintact cells with a loose-fitting pestle in a glass Dounce (10-20 strokes) prior to washing.

Preparation of semiintact cells for study of vesicular trafficking in vitro

Publisher Summary This chapter focuses on techniques used in the laboratory to reconstitute endoplasmic reticulum (ER)-to-Golgi transport in vitro, using adherent tissue culture cells that have been rendered semiintact by gently scraping them from the dish after hypotonic swelling. This technique is discussed in the context of other protocols that allow the investigator to perforate both adherent and nonadherent cells. The ER-to-Golgi transport assay is based on the observation that individual N-linked oligosaccharide-processing enzymes reside in distinct subcellular compartments.Potential markers include endogenous proteins, other viral glycoproteins, and proteins acquired through transfection. Moreover, because both the interior and exterior membranes of the cell are jointly accessible to a wide range of reagents and macromolecules, semiintact cells may provide a useful model system for the study of a broad range of problems in cell biology, including signal transduction, organization of the cytoskeletal matrix, and gene activation.

G Protein Regulation of Vesicular Transport Through the Exocytic Pathway

A central problem in cell biology is to understand the molecular basis for the transport of itinerant soluble and membrane-associated macromolecules through multiple compartments of the exocytic and endocytic pathways of eukaryotic cells. Transport involves a multiplicity of components which facilitate the formation of coat protein complexes leading to vesicle budding, and protein complexes involved in vesicle targeting and fusion to downstream compartments. Many gene products involved have been identified through genetic studies in yeast (reviewed in Hicke and Schekman, 1990) and through biochemical approaches (reviewed in Balch, 1990b; Mellman and Simon, 1992; Rothman, 1992), although an understanding of the enyzmological mechanisms involved are largely unknown.