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

Experimental Approaches for Investigating Disulfide-Based Redox Relays in Cells

“…Protein identification experiments revealed that the yeast cytosolic thioredoxin Trx2 was principally cross-linked via its active site cysteines to several major redox partners (including the thioredoxin reductase Trr1, the peroxiredoxins Tsa1 and Tsa2, the methionine sulfoxide reductase Ykg9, and the sulfiredoxin Srx1). , In other cases, some thioredoxin substrates did not show appreciable cross-linking to thioredoxin in our studies, instead forming internal cross-links between redox centers or undergoing conjugation to GSH . Subsequently, we have used this approach to provide a qualitative assessment of binding interactions between redox partners like Tsa1 and Trx2 in yeast cells. , While most of our work has been performed in baker’s yeast, other groups have employed similar cross-linking approaches with either nitrogen mustard or DVSF to trap interactions between mammalian thioredoxin and thioredoxin reductase. , More recently, this experimental strategy has been applied to identify the interaction partners of protein disulfide isomerases in both baker’s yeast and Plasmodium falciparum , which suggests the possibility of numerous “redox exchange” events between ER-resident chaperones and putative client proteins that enter the secretory pathway. , On the basis of these findings as a whole, our group has posited that cross-linkers like DVSF provide a potential way of identifying protein pairs that normally function in disulfide exchange reactions.…”

“…181 Subsequently, we have used this approach to provide a qualitative assessment of binding interactions between redox partners like Tsa1 and Trx2 in yeast cells. 182,183 While most of our work has been performed in baker's yeast, other groups have employed similar cross-linking approaches with either nitrogen mustard or DVSF to trap interactions between mammalian thioredoxin and thioredoxin reductase. 184,185 More recently, this experimental strategy has been applied to identify the interaction partners of protein disulfide isomerases in both baker's yeast and Plasmodium falciparum, which suggests the possibility of numerous "redox exchange" events between ERresident chaperones and putative client proteins that enter the secretory pathway.…”