earticle

영어

Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance from bacteria to humans. Protein O-mannosylation in yeast and higher eukaryotes is initiated by protein O-mannosyltransferases (PMTs) that transfer mannose from Dol-P-Man to serine or threonine residues of secretory proteins. We identified and characterized two H. polymorpha PMT1 subfamily genes, HpPMT1-1 and HpPMT1-2, in the thermotolerant methylotrophic yeast Hansenula polymorpha. The HpPMT1-1 and HpPMT1-2 gene encodes two Pmt protein isoforms with an overall protein sequence identity of 52.3 % and 24.6 %, respectively, to Saccharomyces cerevisiae Pmt1p. The analysis of hydropathy profiles predicts the two H. polymorpha Pmt proteins to be integral membrane proteins with multiple transmembrane domains. The promoters of both H. polymorpha genes contain an HpHAC1p binding site, in consistent with their induced expression under UPR condition. Whereas the deletion of HpPMT1-1 resulted in apparent cell growth defects under cell wall stress induction conditions and a temperature sensitive phenotype, any detectable defects were not detected in the HpPMT1-2 deletion. However, the Hppmt1-2 mutant showed a significant sensitivity to the PMT1 inhibitor R3A-1c. Furthermore, analysis of cell wall mannoproteins with lectin blotting indicated significant decrease in O-mannosylation in the Hppmt1-1 mutant, but not in Hppmt1-2, compared to the wild type strain. We constructed a HA epitope-tagged version of HpPmt1-1p and confirmed that this construct was fully functional in vivo and localized at the membrane fraction. Altogether, our data support a major role of HpPMT1-1 in protein O-mannosylation and a redundant function of HpPMT1-2.