Open AccessCloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis
Author(s) -
Shin Kasahara,
Hisafumi Yamada,
Toshiyuki Mio,
Yasuhiko Shiratori,
Chikara Miyamoto,
Tomio Yabe,
Tasuku Nakajima,
Eiji Ichishima,
Yasuhiro Furuichi
Publication year - 1994
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.176.5.1488-1499.1994
Subject(s) - biology , open reading frame , saccharomyces cerevisiae , gene , peptide sequence , biochemistry , microbiology and biotechnology
A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S. cerevisiae genomic library. This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame. The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites. The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively. Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein. Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S. cerevisiae genome, and the transcriptional level of HKR1 is extremely low. Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene. Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin. Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.