SULFHYDRYL MODIFICATION OF THE YEAST WBP1P INHIBITS OLIGOSACCHARYL TRANSFERASE-ACTIVITY

Chemical labeling of the multimeric Saccharomyces cerevisiae oligosacc haryl transferase indicates that the 48 kDa Wbp1p subunit is an integr al component of the catalytically active enzyme. The enzyme was purifi ed following chromatography on concanavalin A agarose, heparin agarose , Q-Sepharose, and hydroxyapatite media. The enzyme activity copurifie d with a tetrameric complex of polypeptide subunits. Two of the subuni ts have been identified as the yeast proteins Wbp1p and Swp1p by amino -terminal residue sequencing. A third subunit was identified as a vari ably glycosylated polypeptide near 64 kDa; preliminary amino acid sequ encing showed no identity to known yeast proteins. Modification of a c ysteine residue by the reagent methyl methanethiolsulfonate (MMTS) cau sed time-dependent and concentration-dependent inactivation of the enz yme. To identify the modified subunit of the transferase complex, the labeling reagent S-[(N-biotinoylamino)ethyl] methanethiolsulfonate (BM TS) was synthesized. Like MMTS, BMTS inactivated the oligosaccharyl tr ansferase in a time-dependent manner. Additionally, incubation with th e substrate (dolichylpyrophosphoryl)-N,N'-diacetylchitobiose [Dol-PP(G lcNAc)(2)] protected the enzyme from BMTS inactivation. When the purif ied enzyme complex was incubated with BMTS, Wbp1p alone was specifical ly labeled, thereby associating this subunit with catalysis and the bi nding of the dolichylpyrophosphoryl oligosaccharide substrate in the t ransferase reaction.