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

The deletion of the protein mannosyltransferase 1 gene (PMT1) of Sacch aromyces cerevisiae results in viable cells, O-Mannosylation of protei ns is reduced to about half of the value in comparison to wild-type ce lls. In order to distinguish between the the PMT1 gene product (= Pmt1 p) 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 t he residual enzyme activity for the hexapeptide YNPTSV is 7 times high er than that of Pmt1p and shows a clear preference for the seryl resid ue. Differences in substrate affinities as well as in seryl/threonyl p reference between the two enzymes, however, depend on the specific seq uence of the peptides used in the enzyme assay, The new enzyme activit y shows a significantly lower thermal stability as compared to Pmt1p,