Function of the C-terminal domain of Hkr1, a signaling mucin of Saccharomyces cerevisiae in the context of bud site selection and resistance to the cell wall integrity disruptor HM-1 killer toxin Toshihiro Kondo, Ukyo Suzuki and Shin Kasahara* Food Microbiology Unit, School of Food & Agricultural Sciences, Miyagi University, Sendai, JAPAN *Correspondence; kasahara@myu.ac.jp 1886B Fig. 1 The killer toxin called HM-1 produced by L. mrakii kills some other yeasts such as S. cerevisiae. See the clear zone around growing L. mrakii. HM-1 is known as a cell wall integrity disruptor for sensitive yeasts, also inhibits cell wall b-glucan synthesis. HM-1 is a relatively small protein, consists of 88 amino acids and is very stable at high temperature (over 100℃), high and low pH (4 to 11). by Ryotaro Ozawa Lindnera mrakii (syn. Williopsis saturnus var. mrakii or Hansenula mrakii) produces a proteinous killer toxin called HM-1 which kills sensitive yeasts including Saccharomyces cerevisiae. HKR1 (Hansenula mrakii killer toxin-resistant gene 1) was originally isolated from the genome of S. cerevisiae as a gene whose overexpression overcame the cytocidal effect of HM-1. The gene product Hkr1 is a large, highly glycosylated mucin-like type I transmembrane protein containing an N- terminal signal peptide sequence, Ser/Thr-rich repetitive sequences and a putative transmembrane domain. Calcium binding EF hand and leucine zipper motives were found in its cytoplasmic tail. It also functions as an osmosensor in the high osmolarity glycerol (HOG) MAP kinase pathway. We previously reported that only the partial sequence of Hkr1 endowed HM-1 resistance to the cells, then first in this study, the minimum sequence of Hkr1 required for HM-1 resistance was determined by serial deletions. Apparently the extracellular HMH (Hkr1-Msb2 Homology) domain in addition to the cytoplasmic tail was indispensable for HM-1 resistance. Also we observed that the cells overexpressing partial HKR1 showed altered budding patterns. The haploid S. cerevisiae cells mainly select bud sites in an axial pattern, but bipolar and randomized patterns were often observed in the presence of HM-1. The mutant cells lacking the cytoplasmic part of Hkr1 showed an aberrant budding pattern, too. It is well studied that a series of BUD gene products, Bud1/Rsr1, Bud2, Bud5 and so forth are required for proper bud-site selection in S. cerevisiae. Since both overexpression and disruption of HKR1 gave rise to abnormal budding patterns, Hkr1 might regulate budding coordinately with those proteins, or possibly interacting with some other factors. Moreover, we found that the budding pattern of haploid S. cerevisiae cells grown under the influence of HM-1 was also affected, suggesting that HM-1 perturbed the bud-site selection process. HM-1 has been studied as a cell wall integrity disruptor and believed to inhibit the synthesis of cell wall polysaccharides, but we now postulate that it may target other cellular events such as bud site selection and cell polarity regulation. Our observations could provide important pieces of information to understand the mechanism of the cytotoxicity of HM-1 and the function of Hkr1, especially its cytoplasmic domain in bud site selection as well as cell wall integrity of S. cerevisiae. vector only overexpression of “A” driven by PGAL1 − ＋ − ＋ − ＋ 100 (%) 50 HM-1 killer toxin carbon source monopolar other (deformed) overexpression of “A” driven by PADH1 1120 SFGYSSSSISSIKLSKETIPASKSVSNTQERITSFTSTLRANSQSEKSEGRNSVGSLQSSHISSNPSLST 1190 NTKVDSKSLSRKVSKTMGENGEETGLTTTKTQYKSSSETSGSYSRSFTKISIGPATTAVQTQASTNSVFT 1260 APALSTYPTTPYPSPNSYAWLPTAIIVESSETGPTTASFNPSITGSLPNAIEPAVAVSEPINHTLITIGF 1330 TAALNYVFLVQNPLSSAQIFNFLPLVLKYPFSNTSSELDNSIGELSTFILSYRSGSSTTTLSPKSISSLS 1400 VVKKKKNQQKKNATKSTEDLHPPQVDTSSIAVKKIVPMVDSSKAYIVSVAEVYFPTEAVTYLQQLILDEN 1470 STLYSNPQTPLRSLAGLIDSGIPLGGLTLYGSGDGGYVPSLTSSSVLDSSKGNSQNIDGTYKYGALDDFI 1540 NSFTDSASAGMYAVKIIIFLIVLTIGVLLWLFVAFFAFRHRNILLKRHPRNCIGKSLNNE