Are There Two Protein Disulfide Isomerases in the ER?

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The general aim of this research is the isolation and characterization of components of the endoplasmic reticulum (ER) that are involved in the processing and modification of newly synthesized proteins. This proposal focuses specifically on the components of the ER that may mediate the formation of the correct disulfide bonds during the early stages of protein folding and assembly. The luminal microsomal enzyme protein disulfide isomerase (PDI) has long been the best candidate to carry out this function. Recently, however, a newly described luminal microsomal protein, ERp72, has been cloned in our laboratory. ERp72 has the striking property of possessing three repeats of the amino acid sequences that are thought to be the active site sequences of PDI. Accordingly, the research described in this proposal is designed to provide new insights into the possible functional relationship between ERp72 and PDI. The first specific aim of this proposal is to determine directly whether ERp72 can act as a protein disulfide isomerase. Full- length expressible cDNA clones encoding ERp72 and PDI will be expressed in eukaryotic and bacterial cells and the expressed protein will be assayed for PDI activity directly. The second specific aim of this proposal is to obtain insights into the functional relationship between ERp72 and PDI by investigating and comparing important biological parameters of the two proteins. The relative expression of the two proteins in various tissues will be determined. In addition, the mechanism of ER localization of the two proteins and their protein "neighborhoods" in the ER will be elucidated. The approaches described n this proposal should help us elucidate the way in which the environment in the ER helps to mediate the processes of protein folding and disulfide bond formation. The results of our studies may increase our ability to engineer cells that would be extremely efficient in the synthesis, folding, processing, and transport of cloned protein of potential commercial and therapeutic value.