CLONING AND CHARACTERIZATION OF THE ALG3 GENE OF SACCHAROMYCES-CEREVISIAE

The Saccharomyces cerevisiae alg3-1 mutant is described as defective i n the biosynthesis of dolichol-linked oligosaccharides (Huffaker and R obbins, Proc, Nad Acad, Sci, USA, 80, 7466-7470, 1983), Man(5)GlcNAc(2 )-PP-Dol accumulates in alg3 cells and Endo H resistant carbohydrates are transferred to protein by the oligosaccharyltransferase complex. I n this study, we describe the cloning of the ALG3 locus by complementa tion of the temperature sensitive growth defect of the alg3 stt3 doubl e mutant. The isolated ALG3 gene complements both the defect in the bi osynthesis of lipid-linked oligosaccharides of the alg3-mutant and the under-glycosylation of secretory proteins, The inactivation of the no nessential ALG3 gene results in the accumulation of lipid-linked Man(5 )GlcNAc(2) and protein-bound carbohydrates which are completely Endo H resistant, The ALG3 locus encodes a potential ER-transmembrane protei n of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequenc e.