A NEW DOL-P-MAN-PROTEIN O-D-MANNOSYLTRANSFERASE ACTIVITY FROM SACCHAROMYCES-CEREVISIAE

The deletion of the protein mannosyltransferase 1 gene (PMT1) of Saccharomyces cerevisiae results in viable cells, O-Mannosylation of proteins is reduced to about half of the value in comparison to wild-type cells. In order to distinguish between the the PMT1 gene product (= Pmt1p) and residual transferase activity, an in vitro assay to measure Dol-P-Man:protein mannosyltransferase activity in cells deleted for PMT1 has been developed. The transferase activity of these cells exhibits a pH optimum of 6.5 as compared to pH 7.5 for Pmt1p. The K-m value of the residual enzyme activity for the hexapeptide YNPTSV is 7 times higher than that of Pmt1p and shows a clear preference for the seryl residue. Differences in substrate affinities as well as in seryl/threonyl preference between the two enzymes, however, depend on the specific sequence of the peptides used in the enzyme assay, The new enzyme activity shows a significantly lower thermal stability as compared to Pmt1p,