Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

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We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.", "claims": [{"id": "claim-target-result", "statement": "High-level protein secretion causes global changes in protein expression levels, with precursor availability and membrane composition limiting secretion in Schizosaccharomyces pombe.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:27477394", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast."}, {"asset": "source:pmid:27477394", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation."}, {"asset": "source:pmid:27477394", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "High-level protein secretion causes global changes in protein expression levels, with precursor availability and membrane composition limiting secretion in Schizosaccharomyces pombe.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:27477394"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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