Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae.
Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal 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 each was fused with the structural gene for a reporter protein consisting of a secretion signal, alpha-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of alpha-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.
Hamada
K
K
Department of Mycology, Nippon Roche Research Center, Kanagawa, Japan.
Fukuchi
S
S
Arisawa
M
M
Baba
M
M
Kitada
K
K
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017261:Glycosylphosphatidylinositols