Molecular characterization of a novel protein disulfide isomerase in carrot.

A protein disulfide isomerase (PDI) coding sequence was cloned from a cDNA library derived from carrot (Daucus carota L.) somatic embryos. The cDNA is 2060 bp in length and encodes for a protein of 581 amino acids and molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic reticulum-retention signal at its C-terminus, which is also found in PDIs in plants and animals. Although between the carrot protein and other plant PDIs there is only about 30% identity, the active site regions are almost identical. The corresponding mRNA was found in varying amounts, in all tissues investigated. A recombinant protein expressed from the carrot cDNA clone effectively catalyzed both glutathione-insulin transhydrogenation and the oxidative renaturation of denatured RNase A. These results suggest that the protein coded for by the carrot gene is a novel member of the PDI family in plants. We therefore designated this novel carrot gene PDIL1. The protein expressed by the PDIL1 cDNA sequence had a highly acidic stretch at its N-terminal region (no such domain exists in known plant PDIs), and was located far from known plant PDIs on a maximum likelihood tree. The PDIL1 gene, together with closely-related genes identified in Arabidopsis and tomato, was suggested to belong to a novel subfamily of PDIs.