MEMBRANE TOPOLOGY AND BIOSYNTHESIS OF GLYCOSAMINOGLYCANS

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The long term goals of this research proposal are (1) to understand the role of the membranes of the endoplasmic reticulum and Golgi apparatus (GA) in mechanisms of biosynthesis of glycosaminoglycans, (2) to establish whether the putative translocator protein for Adenosine 3'-phosphate 5' phosphosulfate (PAPS) located in the GA membrane is coded (at least partially) by DNA sequences in common with the ADP/ATP translocator of the mitochondrial membrane and (3) to understand the relationship between glycosaminoglycans to ascribed functions in vivo such as intercellular recognition, cell adhesion and morphogenesis. Towards achieving these goals the following specific aims will be pursued: Continuation with studies on the mechanism(s) of sulfation in the GA. Attempts will be made to identify, purify and reconstitute into liposomes the putative translocator for PAPS. For this purpose advantage will be taken of the apparent structural similarities between the putative PAPS translocator in the GA membrane and the ADP/ATP translocator in mitochondria; the approaches to be used have been successfully applied for similar aims with this latter translocator. Another aim will be the determination of whether the putative PAPS translocator of the GA membrane and the ADP/ATP translocator of mitochondria have common amino acid sequence regions. A series of studies in vitro are proposed to determine whether translocator activities for UDP-Glucuronic acid and UDP-Xylose can be detected in membranes of rat liver rough endoplasmic reticulum and GA; if successful, attempts will be made to identify, purify, and reconstitute the corresponding translocator proteins with liposomes. Concurrent to the above described studies in vitro on sulfation, attempts will be made to isolate mutant Chinese hamster ovary cells deficient in translocation of PAPS into GA in vivo. For this purpose, radiation suicide of cells with 35S-sulfate will be done, followed by screening of mutants via replica-plating autoradiography, and biochemical analyses.