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1.  Hansenula polymorpha Pmt4p Plays Critical Roles in O-Mannosylation of Surface Membrane Proteins and Participates in Heteromeric Complex Formation 
PLoS ONE  2015;10(7):e0129914.
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.
doi:10.1371/journal.pone.0129914
PMCID: PMC4489896  PMID: 26134523
2.  C-Terminal Truncation of α-COP Affects Functioning of Secretory Organelles and Calcium Homeostasis in Hansenula polymorpha 
Eukaryotic Cell  2004;3(1):52-60.
In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding α-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated α-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of α-COP expression was lethal. The α-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed.
doi:10.1128/EC.3.1.52-60.2004
PMCID: PMC329505  PMID: 14871936
3.  Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha 
A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.
doi:10.1128/AEM.69.8.4448-4454.2003
PMCID: PMC169078  PMID: 12902228
4.  Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum 
Background
Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.
Results
We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.
Conclusions
The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.
doi:10.1186/1471-2199-3-15
PMCID: PMC130179  PMID: 12366865

Results 1-4 (4)