Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

Broader than expected tolerance for substitutions in the WCGPCK catalytic motif of yeast thioredoxin 2.

Thioredoxins constitute a key class of oxidant defense enzymes that facilitate disulfide bond reduction in oxidized substrate proteins. While thioredoxin's WCGPCK active site motif is highly conserved in traditional model organisms, predicted thioredoxins from newly sequenced genomes show variability in this motif, making ascertaining which genes encode functional thioredoxins with robust activity a challenge. To address this problem, we generated a semi-saturation mutagenesis library of approximately 70 thioredoxin variants harboring mutations adjacent to their catalytic cysteines, making substitutions in the Saccharomyces cerevisiae thioredoxin Trx2. Using this library, we determined how such substitutions impact oxidant defense in yeast along with how they influence disulfide reduction and interaction with binding partners in vivo. The majority of thioredoxin variants screened rescued the slow growth phenotype that accompanies deletion of the yeast cytosolic thioredoxins; however, the ability of these mutant proteins to protect against H2O2-mediated toxicity, facilitate disulfide reduction, and interact with redox partners varied widely, depending on the site being mutated and the substitution made. We report that thioredoxin is less tolerant of substitutions at its conserved tryptophan and proline in the active site motif, while it is more amenable to substitutions at the conserved glycine and lysine. Our work highlights a noteworthy plasticity within the active site of this critical oxidant defense enzyme.