The goal is to understand the detailed route and mechanism of transport operating along the secretory path, using rat IgM myeloma and mouse lymphoma cells. The research will focus on the Golgi Complex and make use of the techniques of subcellular fractionation, EM immunocytochemistry and glycoprotein biochemistry. The Golgi Complex is usually identified according to structural and a limited number of histochemical characteristics. To obtain a more extensive set of markers of its vesicle populations and cisternae, monoclonal and polyclonal antibodies will be raised. They will be used to map from the ultrastructure of the myeloma cell onto available Golgi-derived subfractions, thereby making it possible to establish the cytologic origins of the subfractions. These fractions will be compared with myeloma rough microsomes with respect to protein and glycoprotein composition, protein turnover, and permeability toward mono- and divalent cations. The antibodies will also be used to investigate the biosynthesis and transport of membrane proteins of the Golgi Complex and as endocytic tracers to probe the cisternal space of the Golgi cisternae. To study the mechanism(s) of transport, the transport of Ig and distribution of antigenic and histochemical markers will be studied in four conditions known to perturb Ig transport: reduction of temperature to 20 degrees C, inhibition of respiration, treatment with monensin to cause partial Na+ -K+ equilibration, and Ca++ withdrawal. Furthermore, the site of Thy-1 transport arrest will be studied in a set of Thy-1-negative lymphoma mutants. These investigations should serve to evaluate the underlying hypothesis that the Golgi Complex is composed of several structurally and functionally distinct subcompartments which intervene sequentially in transport. Such dissection of the secretory path should point the way toward means for regulating transport.
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