Many biological functions have been attributed to the oligosaccharide chains attached to glycoconjugates. With few exceptions, the biosynthesis of these sugar chains and their subsequent modifications take place during trafficking via the secretary and/or endocytic pathways within the cell. On other hand, these glycosylation changes can be used as convenient signposts for the definition of distinctive compartments in these pathways. On the other hand, the trafficking pathways can be viewed as kinetic events in the stepwise biosynthesis of biologically important glycoconjugates, that eventually are expressed at the cell surface as ligands for adhesion receptors, or as tumor antigens. This project focuses on analyzing the glycosylation machinery in the individual subcompartments, along these pathways particularly as it relates to N-linked oligosaccharides. Each step in the late processing of N-linked oligosaccharides occurs in an intact Golgi stack, and requires a specific sugar nucleotide, the corresponding sugar nucleotide transporter, the correct glycosyltransferase, and an appropriate acceptor. We found that this complex set of interactions can be studied in vitro by adding labelled sugar nucleotides to isolated intact Golgi preparations. This incorporation of label into endogenous acceptors is sensitive to detergent, and resistant to proteases. The label is primarily in N-linked oligo saccharides that can be released with PNGaseF, and structurally characterized. The structural information can be used to make conclusions regarding the organization and function of the Golgi apparatus. We will focus here upon the Golgi apparatus of rat hepatocytes in normal and malignant states. The approach we have developed can then be adapted to study the composition of trafficking intermediates, analyzing the trafficking mechanisms themselves, and the role of various cytosolic factors in transport. A particular advantage is that we are first studying the functions of the Golgi apparatus in completely normal cell-type (the hepatocyte). The resulting information can then be compared and contrasted with that form the counterpart malignant cells (hepatoma cells) and also with a variety of other cell types being studied by other members of this project (e.g. erythroleukemia cells, fibroblast and epithelial carcinoma cells). The approaches taken will include in vitro reconstitution, permeabilized cell system, microinjection of intact cells, and the effects of GTP analogues and specific G-proteins antibodies in each situation. This project relates in many ways to the primary theme of this program the study of membrane trafficking and the role of cytosolic factors in controlling it. In the long run, goal is to understand the normal organization of N- linked glycosylation in the Golgi apparatus, its relationship to intercompartmental trafficking and the changes that occur in malignant disease.
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