Activation of the unfolded protein response in <i>Pichia pastoris</i>
 requires splicing of a <i>HAC1</i>
 mRNA intron and retention of the C-terminal tail of Hac1p

Whyteside, Graham; Nor, Rohana Mat; Alcocer, Marcos; Archer, David B.

doi:10.1016/j.febslet.2011.02.036

Cited by 24 publications

(23 citation statements)

References 34 publications

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“…Environmental factors are presumably responsible for the high level of UPR and ERAD related proteins in the batch phase in which high concentrations of glycerol were employed. Moreover, there are also - though not yet conclusive - indications that P. pastoris may exhibit in general a stronger constitutive or basal induction of the UPR as other yeast independent of the production of aberrant proteins [6,12,13]. Our findings also indicate that IP is a quite stable protein as it has been shown that the native-state stability of a secreted protein is inversely correlated to its UPR and ERAD inducing effect [5].…”

Section: Discussionmentioning

confidence: 56%

“…Simplified, overloading of the secretory pathway is sensed in the ER by binding of KAR2 (or BiP) to folding intermediates or misfolded proteins which outcompete the binding of KAR2 to the ER luminal domain of IRE1 [11-13]. The release of KAR2 from the luminal IRE1 domain leads to conformational changes and autophosphorylation and subsequent activation of the cytosolic IRE1 endoribonuclease domain.…”

Section: Introductionmentioning

confidence: 99%

“…The release of KAR2 from the luminal IRE1 domain leads to conformational changes and autophosphorylation and subsequent activation of the cytosolic IRE1 endoribonuclease domain. This leads to splicing of the IRE1 substrate, HAC1 mRNA, being transformed into the activated form encoding the transcriptional activator of UPR responsive genes, Hac1p [11-13]. …”

Section: Introductionmentioning

confidence: 99%

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Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

2014

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BackgroundPichia pastoris is a popular yeast preferably employed for secretory protein production. Secretion is not always efficient and endoplasmic retention of proteins with aberrant folding properties, or when produced at exaggerated rates, can occur. In these cases production usually leads to an unfolded protein response (UPR) and the induction of the endoplasmic reticulum associated degradation (ERAD). P. pastoris is nowadays also an established host for secretory insulin precursor (IP) production, though little is known about the impact of IP production on the host cell physiology, in particular under industrially relevant production conditions. Here, we evaluate the cellular response to aox1 promoter-controlled, secretory IP production in controlled fed-batch processes using a proteome profiling approach.ResultsCells were first grown in a batch procedure using a defined medium with a high glycerol concentration. After glycerol depletion IP production was initiated by methanol addition which was kept constant through continuous methanol feeding. The most prominent changes of the intracellular proteome after the onset of methanol feeding were related to the enzymes of central carbon metabolism. In particular, the enzymes of the methanol dissimilatory pathway - virtually absent in the glycerol batch phase - dominated the proteome during the methanol fed-batch phase. Unexpectedly, a strong decrease of UPR and ERAD related proteins was also observed during methanol-induced IP production. Compared to non-producing control strains grown under identical conditions the UPR down-regulation was less pronounced indicating that IP production elicits a detectable but non prominent UPR response which is repressed by the general culture condition-dependent UPR down-regulation after the shift from glycerol to methanol.ConclusionsThe passage of IP through the secretory pathway using an optimized IP vector and growing the strain at fed-batch conditions with a high initial glycerol concentration does not impose a significant burden on the secretory machinery even under conditions leading to an extracellular accumulation of ~ 3 g L-1 IP. The glycerol batch pre-induction culture conditions are associated with a high constitutive - recombinant protein production independent - induction of the UPR and ERAD pathways probably preconditioning the cells for effective IP secretion in the methanol fed-batch phase.

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Section: Discussionmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

2014

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“…Unlike in S. cerevisiae , Guerfal et al (2010) reported that HAC1 is constitutively expressed and spliced in P. pastoris under normal growth conditions, which may explain the higher titers of secreted proteins obtainable with this organism. A contradictory observation was reported by Whyteside et al (2011). Un-spliced HAC1 mRNA was detected under normal growth conditions and splicing of HAC1 mRNA was only detected when cells were grown in presence of dithiothreitol (DTT) to activate the UPR.…”

Section: Basic Systems For Cloning and Expression In P Pastorismentioning

confidence: 73%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

819

564

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Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

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“…In contrast, the intron of Pichia pastoris HAC1 mRNA is 322 nt, which is longer than the S. cerevisiae HAC1 mRNA intron (10). Despite the differences in intron length, the HAC1 mRNAs of these yeast and filamentous fungi undergo similar, unconventional splicing reactions to produce functional Hac1 proteins (8,9,11).…”

mentioning

confidence: 83%

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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Activation of the unfolded protein response in Pichia pastoris requires splicing of a HAC1 mRNA intron and retention of the C-terminal tail of Hac1p

Cited by 24 publications

References 34 publications

Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

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

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