SCREENING FOR GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-DEPENDENT CELL-WALL PROTEINS IN SACCHAROMYCES-CEREVISIAE

Open reading frames in the genome of Saccharomyces cerevisiae were scr eened for potential glycosylphosphatidylinositol (GPI)-attached protei ns. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a si gnal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of eac h was fused with the structural gene for a reporter protein consisting of a secretion signal, alpha-galactosidase and a hemagglutinin (HA) e pitope, and examined for the ability to become incorporated into the c ell wall. On this basis, 14 of fusion proteins were classified as GPI- dependent cell wall proteins because cells expressing these fusion pro teins: (i) had high levels of alpha-galactosidase activity on their su rface, (ii) released significant amounts of the fusion proteins from t he membrane on treatment with phosphatidylinositol-specific phospholip ase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative G PI-dependent cell wall proteins, 12 had novel ORFs adjacent to their G PI-attachment signal sequence. Amino acid sequence alignment of the C- terminal sequences of the 12 ORFs, together with those of known cell w all proteins, reveals some sequence similarities among them.