In mammals, secretory and cell-surface proteins are modified with one or more types of carbohydrates that are important for a spectrum of physiological functions. In humans, defects in these carbohydrates lead to a variety of diseases. Asparagine-linked (N-linked), these carbohydrates lead to a variety of diseases. Asparagine-linked (N- linked), glycosylphosphatidylinositol (GPI), and C-mannose carbohydrates compete for a common mammose donor, mannose-P-dolichol (MPD). MPD is a component of the """"""""dolichol pathway"""""""" synthesized enzymatically from dolichol-P and GDP-mannose. Since dolichol-P is limiting in cells, the biosynthetic flux for each class of carbohydrate is balanced to maintain cellular needs. In the current funding period, the P.I. cloned a novel gene, termed Lec35, that is essential for MPD utilization. Lec35 mutants synthesize MPD, but have defects in synthesis of N-glycans, GPIs, and C- mannose. Thus, Lec35 represents a new class of protein with a unique mechanism of action. Both the Lec35 cDNA and gene have been cloned by the P.I., and in vivo and in vitro systems for Lec35 function have been devised. For the next funding period five Aims are proposed: I. Determine whether Lec35 protein is involved in (a) MPD flipping from the cytoplasmic leaflet of the ER to the lumenal leaflet, or (b) elimination of a diffusible inhibitor of MPD-dependent mannosylation. II. Determine the topological arrangement, locations of essential residues, and subcellular localization of Lec35 protein, which is predicted to be a resident ER protein with two membrane spans. III. Determine whether Lec35 protein functions prior to, or during, enzymatic MPD-dependent mannosylation. IV. Determine whether the Lec35 protein interacts with other proteins. V. Measure the selectivity of the existing Lec35 cDNA for mannose-P-dolichol over glucose-P-dolichol (GPD), and isolate a cDNA for a GPD-selective form of Lec35.
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