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Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
Published on: November 11, 2014
Functional and molecular characterization of novel Hansenula polymorpha genes, HpPMT5 and HpPMT6, encoding protein
Hyunah Kim1, Hye Yun Moon, Dong-Jik Lee
1Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea.
Abstract:
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.
Insights
The study characterizes two novel protein O-mannosyltransferases, HpPmt5p and HpPmt6p, in Hansenula polymorpha. Their functions are minor but crucial for cell wall integrity and stress resistance when HpPmt1p is absent.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Hansenula polymorpha possesses five PMT genes encoding protein O-mannosyltransferases.
- HpPMT5 and HpPMT6 represent novel PMT1 and PMT2 subfamily members.
Purpose of the Study:
- Systematically characterize the functions of HpPMT5 and HpPMT6.
- Investigate their roles in O-mannosylation, cell wall integrity, and stress resistance.
Main Methods:
- Generation and analysis of single (Hppmt5Δ, Hppmt6Δ) and double (Hppmt1pmt5Δ, Hppmt1pmt6Δ) mutants.
- Assessing sensitivity to Pmt1p inhibitor and cell wall-disrupting reagents.
- Monitoring MAP kinase phosphorylation (HpMpk1p, HpHog1p) and O-mannosylation of HpWsc1p and HpMid2p.
Main Results:
- Single mutants showed no growth defects but increased sensitivity to a Pmt1p inhibitor.
- Double mutants exhibited increased susceptibility to cell wall-damaging agents.
- O-mannosylation of HpWsc1p and HpMid2p was severely impaired in double mutants.
- MAP kinase phosphorylation was induced in double mutants, indicating cell wall stress.
Conclusions:
- HpPmt5p and HpPmt6p play minor roles in O-mannosylation individually.
- Their functions become critical in the absence of HpPmt1p for maintaining cell wall integrity and stress resistance.
- These findings highlight the complex interplay of PMT enzymes in H. polymorpha.
