Molecular Cloning of CWP1: A Gene Encoding a Saccharomyces cerevisiae Cell Wall Protein Solubilized with Rarobacter faecitabidus Protease I

Abstract

A yeast cell wall glycoprotein with a molecular weight of 40, 000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene. The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI. This gene encoded a serine-rich protein of 239 amino acids with a molecular weight of 24, 267. The presence of hydrophobic sequences in the N-and C-termini of the CWP1 protein suggests that it is secreted as a glycosylphosphatidylinositol-anchored protein and is subsequently integrated into the cell wall. Since a gene disruption experiment showed no growth defect, the CWP1 gene is not essential for growth. Mutant CWP1 protein deficient in the C-terminal hydrophobic sequence was secreted into the culture medium, not anchored to the cell wall, thereby indicating that this hydrophobic sequence plays a crucial role in anchoring to the cell wall. Homology between the CWP1 protein and TIP1 family of cold shock proteins suggests that they belong to a new family of cell wall proteins

cell wall protein, gene cloning, glycosylphosphatidylinositol-anchored protein, Rarobacter faecitabidus protease I, Saccharomyces cerevisiae.