Michael O. Agaphonov

Mutation of the homologue of GDP‐mannose pyrophosphorylase alters cell wall structure, protein glycosylation and secretion in Hansenula polymorpha

A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP‐mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP‐mannose, such as cell wall fragility and defects in N‐ and O‐glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum‐associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177. Copyright

Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1

Plasmids with different selectable markers were constructed and used to transform the Hansenula polymorpha strain DL1. It was shown that, depending on the host mutant strain, the use of these plasmids enables rapid selection of transformants with plasmids integrated in low (1–2), moderate (6–9) or high (up to 100) copy numbers. The vectors and mutants described are potentially useful for the construction of efficient producers of heterologous proteins in H. polymorpha. Copyright

Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum

BackgroundSecretion 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.ResultsWe 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.ConclusionsThe 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.

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin‐resistant transformants in Escherichia coli and G418‐resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde‐3‐phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2–2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes. Copyright

Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha.

Human urokinase‐type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein‐O‐mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O‐glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C‐terminal KDEL retention signal. The GenBank Accession No. for the H. polymorpha PMT1 sequence is AY701415. Copyright

Sequences of Saccharomyces cerevisiae 2 μm DNA improving plasmid partitioning in Hansenula polymorpha

Insertion of the HindIII‐PstI fragment of Saccharomyces cerevisiae 2 μm DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 μm DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 μm STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24–28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region.

C-Terminal Truncation of α-COP Affects Functioning of Secretory Organelles and Calcium Homeostasis in Hansenula polymorpha

ABSTRACT 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.

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

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.

Inactivation of Pmc1 vacuolar Ca(2+) ATPase causes G(2) cell cycle delay in Hansenula polymorpha.

The vacuolar Ca2+ ATPase Pmc1 is involved in maintenance of a low Ca2+ concentration in cytosol in yeast cells. Here we observed that increase of Ca2+ cytosolic concentration in yeast Hansenula polymorpha due to inactivation of Pmc1 resulted in sensitivity to sodium dodecyl sulfate (SDS). To elucidate the mechanisms of the observed effect, a screening for mutations suppressing SDS sensitivity of the H. polymorpha pmc1 mutant was performed. As a result, three genes were identified. Two of them, designated as their Saccharomyces cerevisiae orthologs CCH1 and HOG1 encoded the plasma membrane voltage-gated high-affinity calcium channel and the MAP kinase involved in osmoregulation, respectively. The third gene, designated as WEE1, coded for the ortholog of Wee1/Swe1 kinase involved in cell cycle regulation by inhibiting of the G2/M transition. Detailed analysis of this mutant demonstrated that suppression of pmc1 SDS sensitivity by the wee1 mutation depended on an accompanying chromosomal rearrangement, whereas inactivation of WEE1 in the absence of this rearrangement caused SDS sensitivity. Expression of a chimeric protein containing an N-terminal portion of Wee1 in the pmc1 mutant led to abnormal morphology characteristic of G2 delay. Our data indicate that cytosolic Ca2+ rise causes SDS sensitivity in H. polymorpha through the activation of the Wee1 kinase, which is mediated by the Hog1 kinase. Wee1 has a dual role in the manifestation of SDS sensitivity in the H. polymorpha pmc1 mutant. Mechanisms of influence of the obtained mutations on the G2/M transition are discussed.

Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha.

ABSTRACT 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.