Functional and molecular characterization of novel Hansenula polymorpha genes, HpPMT5 and HpPMT6, encoding protein O-mannosyltransferases

• We characterize systematically two novel Hansenula polymorpha genes, HpPMT5 and HpPMT6.
• Mutation of these genes in the absence of HpPMT1 generates apparent defects in cell wall integrity.
• O-mannosylation of some proteins was defected in the Hppmt1pmt5Δ and Hppmt1pmt6Δ double mutants.
• Minor interaction between HpPmt5p and HpPmt2p was detected only in the absence of HpPmt1p.
• HpPmt5p and HpPmt6p become evident upon the loss of HpPmt1p for protein O-mannosylation.

The genome of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologues (HpPMT1, HpPMT2, HpPMT4, HpPMT5, and HpPMT6) encoding protein O-mannosyltransferases. Here, we report on the systematic characterization of HpPMT5 and HpPMT6, encoding novel PMT1 and PMT2 subfamily members, respectively. Although no apparent growth defects were detected in the Hppmt5Δ and Hppmt6Δ single mutants, the single mutants showed dramatic sensitivity to the Pmt1p inhibitor, and the Hppmt1pmt5Δ and Hppmt1pmt6Δ double mutants displayed increased susceptibility to cell wall-disturbing reagents. Activation of the cell wall integrity signaling pathway in the double mutant strains was further indicated by the markedly induced phosphorylation of MAP kinases, such as HpMpk1p and HpHog1p. Noticeably, O-mannosylation of the surface glycoproteins HpWsc1p and HpMid2p became severely defective only in the double mutants, supporting the involvement of HpPmt5p and HpPmt6p in O-mannosylation of these sensor proteins. On the other hand, co-immunoprecipitation experiments revealed only marginal interaction between HpPmt5p and HpPmt2p, even in the absence of HpPmt1p. Taken together, our results suggest that the functions of HpPmt5p and HpPmt6p are minor but become crucial upon the loss of HpPmt1p for protein O-mannosylation, which is essential for cell growth, cell wall integrity, and stress resistance in H. polymorpha.