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Trichoderma reesei has a naturally high capacity for protein secretion and is currently employed for industrial production of a range of enzymes and recombinant gene products for a variety of biotechnological applications. A major limitation for the use of T. reesei as a universal production host is that industrial-scale production of heterologous proteins often results in lower yields than those achieved from native proteins. One reason for the low secretion yields of heterologous proteins is their improper folding and consequent elimination from the cell by the protein quality control mechanisms mediated by the unfolded protein response and the ER-associated degradation. Proteasome plays an important role in protein quality control by degradation the misfolded or aberrant proteins. In the current study three different mutant versions of the main secreted protein, cellobiohydrolase I (CBHI) tagged with the fluorescent protein Venus, were produced in T. reesei and their effects on physiology and gene expression were explored. The transcriptional response of the fungal hyphae was determined by CustomArrayTM 12K slides at three different time points. Potential interaction between the mutant CBHIs and the fungal proteasome was studied by fluorescence and the immunoelectron microscopy. -- A new rapid purification method for the fungal proteasome was developed during this study followed by separation of the proteasome subunit proteins by 2DE. Several proteasome interacting proteins (PIPs) were also identified. The purified 26S proteasome was visualised by transmission electron microscopy. The three mutant CBHI strains differed in terms of protein production and CBHI enzyme activity, although there were similarities between them showing a 'pulsing'-phenomenon both in protein secretion and transcription of the CBHI mRNA. Interestingly only one of the mutant CBHI strains could secrete the Venus-tagged fusion protein into the culture medium. -- The genome wide transcriptional study showed that two mutations in the cbh1 core gene did not cause UPR or ERAD activation, even though physiological signs of the stress were evident. Four and five mutations in the cbh1 core gene lead to expression changes in genes related to UPR and ERAD pathways and the physiological indications of stress were also seen under the light microscope. A new finding was up-regulation of a group of genes involved in 'ribosome structure and synthesis' in all mutant CBHI strains. In previous studies, secretion stress has been applied to fungal hyphae by drugs such as dithiothreitol (DTT) or tunicamycin, which seem to result in a different feedback to the protein translation machinery. -- Fluorescence and immunoelectron microscopy studies supported the microarray results indicating that four mutations in the cbh1 core gene lead to the interaction of the mutant CBHI with the 20S proteasome and at least partial retention of the mutant CBHI protein in the fungal hyphae. |
