title: Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30.
abstract: Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Macquarie University, NSW 2109, Australia. liisa.kautto@mq.edu.au
authors.forename: Liisa
authors.initials: L
authors.lastname: Kautto
authors.forename: Jasmine
authors.initials: J
authors.lastname: Grinyer
authors.forename: Ian
authors.initials: I
authors.lastname: Paulsen
authors.forename: Sasha
authors.initials: S
authors.lastname: Tetu
authors.forename: Aneesh
authors.initials: A
authors.lastname: Pillai
authors.forename: Swapneel
authors.initials: S
authors.lastname: Pardiwalla
authors.forename: Ugur
authors.initials: U
authors.lastname: Sezerman
authors.forename: Gunseli Bayaram
authors.initials: GB
authors.lastname: Akcapinar
authors.forename: Peter
authors.initials: P
authors.lastname: Bergquist
authors.forename: Junior
authors.initials: J
authors.lastname: Te'o
authors.forename: Helena
authors.initials: H
authors.lastname: Nevalainen
mesh_terms: D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation / Q000187:drug effects / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D025301:Hyphae / Q000187:drug effects / Q000201:enzymology; D007976:Leupeptins / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D013312:Stress, Physiological / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects; D014242:Trichoderma / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*
chemical_list: D005656:Fungal Proteins; D007976:Leupeptins; D050505:Mutant Proteins; D061988:Proteasome Inhibitors; D043366:Cellulose 1,4-beta-Cellobiosidase; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde