ASPARTIC-ACID-252 AND ASPARAGINE-185 ARE ESSENTIAL FOR ACTIVITY OF LIPID N-ACETYLGLUCOSAMINYLPHOSPHATE TRANSFERASE

A key step in the assembly of oligosaccharide-lipid intermediates in N -linked glycosylation is the transfer of N-acetylglucosamine 1-phospha te to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosam inyl:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase (L -G1PT). Comparison of the amino acid sequences of L-G1PT from five div erse species showed 75 amino acids identical in all five proteins, Usi ng site-directed mutagenesis, we analyzed the importance of a number o f these conserved residues to the enzymatic activity of L-G1PT using a plasmid shuffling procedure in Schizosaccharomyces pombe. S.pombe cel ls containing a chromosomal deletion of the essential gpt(+) gene are rescued by a plasmid containing the S.pombe gpt open reading frame, Re placement of that plasmid by a plasmid encoding a mutated hamster L-G1 PT cDNA sequence indicated that the mutated protein provided sufficien t enzyme activity to permit cell growth, Mutations of aspartic acid 25 2 and asparagine 185 did not allow plasmid shuffling, indicating these residues were essential for activity, A combination of mutations at a sparagine 182 and tryptophan 122 did not allow plasmid shuffling, alth ough the single mutations did, Overexpression of the mutant proteins i n S.pombe conferred tunicamycin (TM) resistance, indicating that the m utant proteins had a conformation necessary for binding TM, a substrat e analog, The mutant proteins were also detected in Western blots and were correctly localized to the membrane fractions, However, the overe xpressed proteins did not increase the endogenous level of enzymatic a ctivity in these cells, indicating they were enzymatically inactive.