Overexpression of the transcription factor Yap1 modifiesintracellular redox conditions and enhances recombinant proteinsecretion

摘要

Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolicredox state to the levels of the wild type. These alterations are also reflectedby increased levels of oxidized intracellular glutathione (GSSG) in theYAP1 co-overexpressing strain. Taken together, these dataindicate a strong impact of intracellular redox balance on the secretion of(recombinant) proteins without affecting protein folding per se. Re-establishingsuitable redox conditions by tuning the antioxidant capacity of the cell reducesmetabolic load and cell stress caused by high oxidative protein folding load,thereby increasing the secretion capacity.