CLONING OF THE SACCHAROMYCES-CEREVISIAE GENE WHOSE OVEREXPRESSION OVERCOMES THE EFFECTS OF HM-1 KILLER TOXIN, WHICH INHIBITS BETA-GLUCAN SYNTHESIS

A gene whose overexpression can endow Saccharomyces cerevisiae cells w ith resistance to HM-1 killer toxin was cloned from an S. cerevisiae g enomic library. This gene, designated HKR1 (Hansenula mrakii killer to xin-resistant gene 1), contains a 5.4-kb open reading frame. The predi cted amino acid sequence of the protein specified by HKR1 indicates th at the protein consists of 1,802 amino acids and is very rich in serin e and threonine, which could serve as O-glycosylation sites. The prote in also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and tran smembrane domain, respectively. Hkr1p is found to contain an EF hand m otif of the calcium-binding consensus sequence in the C-terminal cytop lasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycos ylated type I membrane protein. Southern and Northern (RNA) analyses d emonstrated that there is a single copy of the HKR1 gene in the S. cer evisiae genome, and the transcriptional level of HKR1 is extremely low . Gene disruption followed by tetrad analysis showed that HKR1 is an e ssential gene. Overexpression of the truncated HKR1 encoding the C-ter minal 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 acti vity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.