Trx1 kinetic trapping is mediated by specific protein–protein interactions
To further investigate whether the capture of proteins by Trx1(CSAAA) is Trx-specific, we directly compared Trx1(CSAAA) with the corresponding trapping mutant of another member of the Trx superfamily, glutaredoxin-1 Grx1(CSAAA) (CSAAA annotates the identity of residues 22, 25, 7, 78 and 82). Grx1, like Trx1, uses its active site thiol to act as a disulfide reductase in the cytosolic environment. However, in contrast to Trx1, Grx1 is specialized in the recognition and reduction of protein–glutathione mixed disulfide bonds and forms mixed disulfide intermediates with glutathione rather than with proteins (Yang et al, 1998; Peltoniemi et al, 2006). As expected, we did not detect trapping of peroxiredoxins (or other Trx1-interacting proteins) by Grx1(CSAAA), neither on silver gels (Figure 1E, lanes 3 and 4) nor by immunoblotting (data not shown). The activity and thiol reactivity of the Grx1 trapping mutant was confirmed in independent experiments (data not shown), thus demonstrating that the mere availability of an active site thiol does not explain the profile of proteins captured by the Trx1 trapping mutant. Instead, our results support the notion that Trx-mediated reducing activity is steered toward distinct target disulfide bonds by specific protein–protein interactions.