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 asparagine residues of nascent polypeptide chains as they are translocated into the lumen of the endoplasmic reticulum. To date, this enzyme has been purified from a number of eukaryotic organisms. Purification of transferase activity has yielded polypeptide complexes of three to six subunits depending on the source organism. Here we present the purification of an affinity-tagged version of the enzyme complex from a membrane protein fraction of the yeast Saccharomyces cerevisiae. A yeast strain was created in which the essential 64-kDa glycoprotein Nlt1p subunit of the oligosaccharyl transferase was modified by the addition of a 22-residue carboxy-terminal affinity tag; the tag included both an 8-residue FLAG epitope and a 6-residue histidine motif. Facile purification of the oligosaccharyl transferase was achieved using affinity chromatography media specific for each segment of the tag. The enzyme was purified as a heteromeric complex of five subunits in agreement with previously reported characterizations of the yeast transferase. Yeast strains bearing affinity-tagged enzyme subunits allow the rapid characterization of native and mutant transferase complexes.
Pathak
R
R
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Imperiali
B
B
D000345:Affinity Labels; D000595:Amino Acid Sequence; D005800:Genes, Fungal; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014166:Transferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000345:Affinity Labels; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase