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.
ASJC Scopus subject areas
- Molecular Biology