We have described that protein-linked, polymannose-type oligosaccharides lacking glucose units are glucosylated directly from UDP-Glc in the lumen of the endoplasmic reticulum (ER) of mammalian, plant, fungal and protozoan cells and that the glucose units are immediately removed, probably by glucosidase II. The long term goal of this application is to find a role for these reactions. The UDP-Glc:glycoprotein glucosyltransferase has been purified to homogeneity from rat liver. Cell-free assays showed that the enzyme glucosylates much more efficiently denatured than native glycoproteins and that this is caused by the interaction of protein determinants exposed in denatured but not in native conformations with the glucosyltransferase. It was suggested that in vivo only unfolded, partially folded and misfolded glycoproteins are glucosylated and that glucosylation stops once glycoproteins adopt their proper tertiary structures. In order to test the possible involvement of the glucosylation reaction in the mechanism by which cells dispose of misfolded structures in the ER or in that by which glycoproteins fold correctly in the same subcellular location we propose: a) to clone and sequence the DNA coding for the UDP-Glc:glycoprotein glucosyltransferase in rat liver and Schizosaccharomyces pombe; b) to disrupt the coding gene in S. pombe and if possible also in mammalian cells and to determine if the absence of the glucosyltransferase modifies the rate of secretion of glycoproteins in those cells. The rate of folding has been recognized to be one of the main pace limiting steps in the transit of proteins from the ER to the ultimate subcellular locations; c) to study if the glucosyltransferase influences the rate of folding of glycoproteins synthesized in a cell-free transcription- translation system; d) to study if the pure enzyme influences the thermal denaturation of glycoproteins; e) as peptides and denatured, deglycosylated glycoproteins were found to inhibit cell-free glucosylation, we propose to synthesize peptides of defined length and random composition, to determine the size, composition and sequence of those showing maximum inhibitory effect, and in this way determine the nature of the domain, exposed in all denatured but not in native glycoproteins, that interacts with the glucosyltransferase.
