Functional and Molecular Characterization of PMT5 and PMT6 genes encoding Protein O-Mannosyltransferases in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

다국어 초록 (Multilingual Abstract)

Protein O-mannosylation is evolutionarily conserved protein modification of fundamental importance, which is mediated by protein O-mannosyltransferases(Pmtproteins). The whole genome information of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologues (HpPMT1, HpPMT2, HpPMT4, HpPMT5, and HpPMT6) encoding Pmt proteins, In this study, we carried out molecular characterization of HpPMT5 and HpPMT6, which show homologies to Saccharomyces cerevisiae PMT1 and PMT2 subfamily members, respectively. Although any detectable growth defects were not detected in the Hppmt5 and Hppmt6 single deletion mutants, they showed significantly increased sensitivity to the PMT1 inhibitor R3A-1c. As expected, the Hppmt1pmt5 and Hppmt1pmt6 double mutants became more susceptible to cell wall disturbing reagents, compared to the single Hppmt1 mutant. Furthermore, O-mannosylation of HpWsc1p and HpMid2p, the cell surface sensors of cell wall integrity-signaling, was significantly defected in the both Hppmt1pmt5 and Hppmt1pmt6 mutants. Interestingly, phosphorylaiton of Mpk1 protein, which results in the stimulation of the cell wall integrity pathway, was more markedly induced in the Hppmt1pmt5 than in Hppmt1 even under normal condition. The membrane fractionation experiment showed that all the HpPmt proteins are localized at the ER/Golgi membrane except HpPmt6p, which was mostly detected as soluble protein. By co-immunoprecipitation experiments, we confirmed the complex formation between HpPmt1p and HpPmt2p, but no interaction between HpPmt5p and HpPmt2p. Altogether, these results support that HpPmt5p and HpPmt6p have redundant functions to significantly compensate the loss of HpPmt1p in protein O-mannosylation that are essential for cell growth, cell wall integrity, and stress resistance of H. polymorpha.