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Molecular and General Genetics MGG

, Volume 258, Issue 1–2, pp 53–59 | Cite as

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

  • K. Hamada
  • S. Fukuchi
  • M. Arisawa
  • M. Baba
  • K. Kitada
ORIGINAL PAPER

Abstract

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, α-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 α-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.

Key wordsSaccharomyces cerevisiae Yeast Cell wall protein GPI-anchored protein 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • K. Hamada
    • 1
  • S. Fukuchi
    • 1
  • M. Arisawa
    • 1
  • M. Baba
    • 1
  • K. Kitada
    • 1
  1. 1.Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura-shi, Kanagawa 247, Japan Fax: +81-467-46-5320; e-mail: kunio.kitada@roche.comJP

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