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.

A strain of the yeast Saccharomyces cerevisiae coding for KHR on the chromosome secreted a toxin that kills sensitive yeasts. The transformants of multicopy vectors carrying the KHR gene could secrete 3-4-fold the killer toxin of the donor strain. This toxic substance was purified 80-fold in specific activity from the culture filtrate by gel filtration and hydrophobic column chromatography. The purified toxin gave a single protein band with molecular mass of 20 kDa on SDS-PAGE and had an isoelectric point of pH 5.3. The toxin had novel killer activity against Candida glabrata and S. cerevisiae, but did not affect bacteria, fungi, or other yeasts.

The KHRgene cloned from a genomic library was on 4.7-kbp DNAfragment and was inserted into YCpGll vector (KHR-YCp) and YEp vector (KHP-YEp). Transformants with KHR-YEpcould secrete 3-4 times as muchkiller toxin into the media as the donor strain. Thecomplete nucleotide sequence of the KHRgene was analyzed. It was found that the KHRgene consisted of 888 bp. It was suggested that this protein was processed before being secreted into the media, because its molecular mass presumedfrom the nucleotide sequence was larger than that of the mature killer toxin. To study the function and the secretion system of KHRkiller toxin in comparison with other killer toxin, the elucidation of its primary structure is necessary. We report here the molecular cloning and the nucleotide se-

Isoamyl acetate, one of the major characteristic aroma compoundsof sake, was synthesized by the condensation of acetyl-CoA and isoamyl alcohol under the action of alcohol acetyltransferase (EC 2.3.1.84) of yeast. The enzyme, which was associated with the cell membrane, was purified about 800-fold. The purified enzyme was very labile at temperatures higher than 10°C and pHs lower than 5.5 but was stable at pH 6.0-7.5. The enzyme was most active at 30°C and pH 7.0. The isoelectric point of this enzymewas 5.5. The molecular weight was estimated to be about 50,000 by gel filtration and SDS polyacrylamide gel electrophoresis.

No abstract