K-043. Specificity of Redox Enzyme Maturation Protein Binding to RR-Containing Peptides of Tat-Dependent Proteins

C. S. Chan, L. Chang, R. J. Turner;
Univ. of Calgary, Calgary, AB, CANADA.

The twin arginine translocase (Tat) system is used by many bacteria and plants to move proteins across the cytoplasmic or thykaloid membrane. Proteins used by this system are pre-folded and contain a conserved SRRxFLK twin arginine (RR-) motif in their N-terminal sequence. Majority of proteins in Escherichia coli that use the Tat pathway are redox enzymes involved in anaerobic respiration and contain cofactors that are inserted prior to translocation. These redox enzymes were also identified to have system-specific chaperones, termed Redox Enzyme Maturation Proteins (REMPs) that may play important roles for cofactor insertion, enzyme maturation, protease protection, and targeting towards the translocon. Binding of some REMPs to the RR-containing region of the Tat substrates has been previously observed. Here we describe the characterization of binding of 10 REMPs towards 16 N-terminal/leader RR-containing peptides of known and predicted Tat-specific redox enzymes. A combination of in vitro and in vivo experiments were investigated and demonstrated that some REMPs were specific to their predicted redox enzyme or enzymes of similar function, whereas others were less specific and bound peptides of unrelated redox enzymes. By comparing the results obtained by in vitro far-Western/overlay to in vivo bacterial two-hybrid (BACTH) experiments, additional interactions were observed by BACTH suggesting that other cellular factors may be involved. The BACTH experiments also showed one additional interaction when determined by enzymatic assay of β-galactosidase from liquid cultures versus by red/white colony observation. Upon performing Western blots against the BACTH constructs of REMPs and peptides from cultures expressing both proteins, it was noticed that the interaction of the two prevents both from being proteolytically degraded. From this study, we demonstrate the specificity of REMP binding to the RR-containing peptides of redox enzymes and show that the interaction of the two has a protection effect against degradation.