Abstract
Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1.