The yeast Williopsis saturnus var. mrakii (syn. Hansenula mrakii) produces a proteinous killer toxin called HM-1, which has a strong cytocidal effect on sensitive yeasts such as Saccharomyces cerevisiae. HKR1 (Hansenula mrakii killer toxin resistant gene 1) was originally identified in the genome of S. cerevisiae as a gene whose overexpression overcame the effect of HM-1. The gene product Hkr1p is a large cell surface protein composed of 1,802 amino acids. It contains a signal peptide sequence at the N-terminus, Ser/Thr-rich mucin-like repetitive sequences and a putative transmembrane domain. Also the calcium binding EF hand motif and the leucine zipper are found in its cytoplasmic tail and the extracellular domain is presumed to be highly glycosylated. It is hence categorized as a transmembrane signaling mucin. Recently it has been reported that Hkr1p and another transmembrane signaling mucin Msb2p act as putative osmosensors in the high osmolarity glycerol (HOG) signaling pathway.
We here report that only short portions of HKR1 endow HM-1 resistance to S. cerevisiae cells. Moreover, even when the reverse strand of HKR1 was transcribed (presumably the expression of the gene was suppressed by the antisense RNA), S. cerevisiae cells acquired resistance to HM-1. Since mutations at the positions of internal ATGs which could act as translation initiation codons in HKR1 did not affect the resistance to HM-1, we speculated that the acquisition of HM-1 resistance by partial HKR1 expression was independent from the translation event, that is, overexpression of Hkr1p was not the case. Also we speculate that the expression of HKR1 was downregulated rather than overexpressed due to transgenic cosuppression when the gene was driven by the GAL1 promoter, then the accumulation of the gene product Hkr1p declined finally. From these observations, we postulate that when the expression level of HKR1 was altered, especially downregulated, the cells acquired resistance to HM-1. Quantifying studies for Hkr1p accumulation using antibodies are anticipated. We also observed budding patterns of those transformants expressing partial HKR1. The cells of the haploid S. cerevisiae A451 strain typically select budding sites axially in a unipolar pattern, but bipolar and randomized budding patterns were observed more frequently under the existence of HM-1. We then found that expression levels of HKR1 affected bud site selection of S. cerevisiae as well.
These observations will provide critical information to understand the mechanism of the cytocidal effect of HM-1 and the function of Hkr1p in the cellular signaling of S. cerevisiae in the context of HM-1 resistance.