PMT1 MANNOSYL TRANSFERASE IS INVOLVED IN CELL-WALL INCORPORATION OF SEVERAL PROTEINS IN SACCHAROMYCES-CEREVISIAE

We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowe d us to identify a cell wall-bound form of Srp1p. We showed that the l ast 30 amino acids of Srp1p, but not the last 16, contain sufficient i nformation to signal glycosyl-phosphatidylinositol anchor attachment a nd subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta 1,6-glucose-containing sidechain. Pmt1 p enzyme is known as a protein-O-mannosyltransferase that initiates th e O-glycosidic chains on proteins. We found that a pmt1 deletion mutan t was highly sensitive to zymolyase and that in this strain the alpha- galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein we re absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phospha tidylinositol anchor in pmt1 cells, and in this mutant strain an alpha -galactosidase-Cwp2 fusion protein was found linked to the cell wall b ut devoid of beta 1,6-glucan side-chain, indicating an alternative mec hanism of cell wall anchorage.