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Hansenula polymorpha Pmt4p Plays Critical Roles in O-Mannosylation of Surface Membrane Proteins and Participates in Heteromeric Complex Formation.

ABSTRACT: O-mannosylation, the addition of mannose to serine and threonine residues of secretory proteins, is a highly conserved post-translational modification found in organisms ranging from bacteria to humans. Here, we report the functional and molecular characterization of the HpPMT4 gene encoding a protein O-mannosyltransferase in the thermotolerant methylotrophic yeast Hansenula polymorpha, an emerging host for the production of therapeutic recombinant proteins. Compared to the deletion of HpPMT1, deletion of another major PMT gene, HpPMT4, resulted in more increased sensitivity to the antibiotic hygromycin B, caffeine, and osmotic stresses, but did not affect the thermotolerance of H. polymorpha. Notably, the deletion of HpPMT4 generated severe defects in glycosylation of the surface sensor proteins HpWsc1p and HpMid2p, with marginal effects on secreted glycoproteins such as chitinase and HpYps1p lacking a GPI anchor. However, despite the severely impaired mannosylation of surface sensor proteins in the Hppmt4? mutant, the phosphorylation of HpMpk1p and HpHog1p still showed a high increase upon treatment with cell wall disturbing agents or high concentrations of salts. The conditional Hppmt1pmt4? double mutant strains displayed severely impaired growth, enlarged cell size, and aberrant cell separation, implying that the loss of HpPMT4 function might be lethal to cells in the absence of HpPmt1p. Moreover, the HpPmt4 protein was found to form not only a homomeric complex but also a heteromeric complex with either HpPmt1p or HpPmt2p. Altogether, our results support the function of HpPmt4p as a key player in O-mannosylation of cell surface proteins and its participation in the formation of heterodimers with other PMT members, besides homodimer formation, in H. polymorpha.

SUBMITTER: Kim H

PROVIDER: S-EPMC4489896 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Hansenula polymorpha Pmt4p Plays Critical Roles in O-Mannosylation of Surface Membrane Proteins and Participates in Heteromeric Complex Formation.

Kim Hyunah H Thak Eun Jung EJ Lee Dong-Jik DJ Agaphonov Michael O MO Kang Hyun Ah HA

PloS one 20150702 7

O-mannosylation, the addition of mannose to serine and threonine residues of secretory proteins, is a highly conserved post-translational modification found in organisms ranging from bacteria to humans. Here, we report the functional and molecular characterization of the HpPMT4 gene encoding a protein O-mannosyltransferase in the thermotolerant methylotrophic yeast Hansenula polymorpha, an emerging host for the production of therapeutic recombinant proteins. Compared to the deletion of HpPMT1, d ...[more]

PMID: 26134523

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Project description:BACKGROUND: Methylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism.In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses. RESULTS: Two hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal beta-oxidation.A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11.Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation. CONCLUSION: Transcriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal beta-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).

Dataset Information

Hansenula polymorpha Pmt4p Plays Critical Roles in O-Mannosylation of Surface Membrane Proteins and Participates in Heteromeric Complex Formation.

Publications

Hansenula polymorpha Pmt4p Plays Critical Roles in O-Mannosylation of Surface Membrane Proteins and Participates in Heteromeric Complex Formation.

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