Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (Ca BP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cy s) respectively and, like PDI, may be involved in the folding of nascent pr oteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can compl ement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI ge ne, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replac ed by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays ac id in vivo pro(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is si gnificantly more active than the second trx box. Furthermore, only about 65 Yb re-activation of denatured reduced RNase AIII could be obtained with Ca BP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions wa s significantly reduced in the presence of either CaBP1 or CaBP2, In contra st with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of den atured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-i ndependent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologu e, and although PDI can bind malfolded proteins such as 'scrambled' ribonuc lease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the gene ral peptide-binding activity attributed to the b' domain of PDI; (3) intera ction of CaBP2 with substrate (RNase AIII) is different from that of PDI an d substrate; and (4) both CaSP2 and CaBP1 may promote oxidative folding by different kinetic pathways.