A DUAL AFFINITY TAG ON THE 64-KDA NLT1P SUBUNIT ALLOWS THE RAPID CHARACTERIZATION OF MUTANT YEAST OLIGOSACCHARYL TRANSFERASE COMPLEXES

Oligosaccharyl transferase catalyzes the glycosylation of selected asp aragine residues of nascent polypeptide chains as they are translocate d into the lumen of the endoplasmic reticulum. To date, this enzyme ha s been purified from a number of eukaryotic organisms. Purification of transferase activity has yielded polypeptide complexes of three to si x subunits depending on the source organism. Here we present the purif ication of an affinity-tagged version of the enzyme complex from a mem brane protein fraction of the yeast Saccharomyces cerevisiae. A yeast strain was created in which the essential 64-kDa glycoprotein Nltlp su bunit of the oligosaccharyl transferase was modified by the addition o f a 22-residue carboxy-terminal affinity tag; the tag included both an 8-residue FLAG epitope and a 6-residue histidine motif. Facile purifi cation of the oligosaccharyl transferase was achieved using affinity c hromatography media specific for each segment of the tag. The enzyme w as purified as a heteromeric complex of five subunits in agreement wit h previously reported characterizations of the yeast transferase. Yeas t strains bearing affinity-tagged enzyme subunits allow the rapid char acterization of native and mutant transferase complexes. (C) 1997 Acad emic Press.