Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast
            <i>Saccharomyces cerevisiae</i>

Valkonen, Mari; Penttilä, Merja; Saloheimo, Markku

doi:10.1128/aem.69.4.2065-2072.2003

Cited by 168 publications

(119 citation statements)

References 41 publications

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“…S2A in the supplemental material), consistent with previous reports that the overexpression of endogenous or heterogeneous active Hac1 proteins severely reduces the rate of S. cerevisiae growth (19,20). However, expression of the HpHAC1 u form rescued the defective growth of the Schac1⌬ mutant subjected to TM stress.…”

Section: Expression Of Hphac1supporting

confidence: 79%

Hansenula polymorpha Hac1p Is Critical to Protein N -Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

Moon

Cheon

Kim

et al. 2015

Appl Environ Microbiol

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dAggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular protective response to ER stress, the unfolded protein response (UPR), which is mediated by a basic leucine zipper (bZIP) transcription factor, Hac1p/Xbp1. In this study, we identified and studied the molecular functions of a HAC1 homolog from the thermotolerant yeast Hansenula polymorpha (HpHAC1). We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose interaction with the 5= untranslated region is responsible for the translational inhibition of the HpHAC1 mRNA. The H. polymorpha hac1-null (Hphac1⌬) mutant strain grew slowly, even under normal growth conditions, and was less thermotolerant than the wild-type (WT) strain. The mutant strain was also more sensitive to cell wall-perturbing agents and to the UPR-inducing agents dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the WT and Hphac1⌬ strains treated with DTT and TM, we identified HpHAC1-dependent core UPR targets, which included genes involved in protein secretion and processing, particularly those required for N-linked protein glycosylation. Notably, different glycosylation and processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the WT and Hphac1⌬ strains. Moreover, overexpression of active HpHac1p significantly increased the N-linked glycosylation efficiency and TM resistance. Collectively, our results suggest that the function of HpHac1p is important not only for UPR induction but also for efficient glycosylation in H. polymorpha. The endoplasmic reticulum (ER) represents the central organelle of the eukaryotic cell in which the crucial steps of protein folding, modification, and selection for transport to other compartments take place. Perturbation of ER homeostasis through, for example, the accumulation of misfolded proteins or defects in the ER membrane leads to the activation of intracellular signaling pathways referred to as the unfolded protein response (UPR), ERassociated protein degradation (ERAD), and the calnexin cycle (CNX) for glycoprotein quality control (1). UPR induces multiple protective cellular events required for proper protein folding and misfolded protein degradation. The underlying regulatory mechanism and modes of action of UPR have been intensively studied in the traditional yeast Saccharomyces cerevisiae (2). UPR is mediated by a master transcription factor, Hac1p, in S. cerevisiae (ScHac1p) (3), and the synthesis of functionally active Hac1p is triggered via nonconventional splicing of the HAC1 mRNA, which is mediated by the endonuclease activity of the ER stress sensor, Ire1p (4). This unusual splicing of HAC1 mRNA induces the translation of HAC1 mRNA by eliminating base pairing between the 5= untranslated region (UTR) and the splicing site (5). The newly synthesized Hac1p is then shuttled into the nucleus and functions as an active basic leucine zipper (bZIP) transcription factor during ER stress. Hac1p binds to a UPR element (UPRE) within the pr...

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Section: Expression Of Hphac1supporting

confidence: 79%

Hansenula polymorpha Hac1p Is Critical to Protein N -Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

Moon

Cheon

Kim

et al. 2015

Appl Environ Microbiol

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“…If a large fraction of the immunotoxin unfolds in the endoplasmic reticulum, the presence of a robust UPR could enhance secretion of the immunotoxin (22). In FIG.…”

Section: Discussionmentioning

confidence: 99%

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

Woo

Liu

Stavrou

et al. 2004

Appl Environ Microbiol

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The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut ؉ ) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and tolerance induction for transplantation. This immunotoxin selectively kills human T cells and is the most efficacious of a variety of anti-T-cell immunotoxins that have been developed (20). The bivalent immunotoxin contains the first 390 amino acid residues of diphtheria toxin (DT) and two tandem sFv molecules that are responsible for binding the immunotoxin to the CD3ε␥ subunit of the T-cell receptor complex on human T cells. The first 390 amino acid residues of DT (DT390) contain the catalytic domain or A chain of DT that inhibits protein synthesis by ADP ribosylation of EF-2 and the translocation domain that translocates the catalytic domain to the cytosol by interactions with cytosolic Hsp90 and thioredoxin reductase (17).The inhibition of protein synthesis by the catalytic domain makes it difficult to produce the toxin-related proteins in most eukaryotic cells (e.g., wild-type CHO cells, insect cells, and yeasts). The use of toxin-resistant eukaryotic cells can overcome the immunotoxin toxicity. However, selection and characterization of toxin-resistant eukaryotic cells are tedious, labor-intensive, ...

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“…Manipulation of UPR has been used as well to improve heterologous protein production. Disruption and overexpression of HAC1 has been shown to lead to a decrease and increase, respectively, in the production of α-amylase in S. cerevisiae (Valkonen et al 2003). However, activation of the UPR as a whole has a great impact on various cellular processes and therefore does not improve our understanding of the contribution of individual components of UPR regulated genes in resolving the ER stress.…”

Section: Discussionmentioning

confidence: 99%

Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption

Ruijter

Frey

2015

Appl Microbiol Biotechnol

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One of the main limitations for heterologous protein production in the yeast Saccharomyces cerevisiae is the protein folding capacity in the endoplasmic reticulum (ER). Accumulation of unfolded proteins triggers the unfolded protein response (UPR), which resolves the stress by increasing the capacity for protein folding and removal of unfolded proteins by the ER associated degradation (ERAD) system. In order to analyse the influence of ERAD on production of a human IgG, we disrupted ERAD at different stages by deletion of the HTM1, YOS9, HRD1, HRD3 or UBC7 gene, with or without a disruption of the UPR by deletion of the IRE1 gene. All deletion strains were viable and did not exhibit a growth phenotype under normal growth conditions. Deletion of HTM1 resulted in a small increase in antibody production, whereas a small decrease in antibody production was observed in the Δhrd1, Δhrd3 and Δubc7 yeast strains, and a stronger decrease in the Δyos9 yeast strain. Deletion of the IRE1 gene had contrasting effects in the ERAD mutants, with a strongly decreased production in wild type cells and partially reversed effects in combination with the Δhtm1 or the Δyos9 deletions. In order to study IgG clearance from the ER, an assay was developed using the inhibitory effect of glucose on the GAL1 promoter that is driving IgG expression. The Δyos9Δire1and Δhtm1Δire1 strains showed a delayed IgG clearance from the cells, showing that removal of components for the generation and recognition of the glycan signal needed for ERAD mediated protein degradation might increase the IgG ER residence time.3

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Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae

Cited by 168 publications

References 41 publications

Hansenula polymorpha Hac1p Is Critical to Protein N -Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

Hansenula polymorpha Hac1p Is Critical to Protein N -Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption

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