PROTEIN STRUCTURE AND FOLDING
Domains b′ and a′ of Protein Disulfide Isomerase Fulfill the Minimum Requirement for Function as a Subunit of Prolyl 4-Hydroxylase: THE N-TERMINAL DOMAINS a AND b ENHANCE THIS FUNCTION AND CAN BE SUBSTITUTED IN PART BY THOSE OF ERp57*

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Protein disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a,b, b′, and a′, plus an acidic C-terminal extension, c. PDI carries out multiple functions, acting as the β subunit in the animal prolyl 4-hydroxylases and in the microsomal triglyceride transfer protein and independently acting as a protein folding catalyst. We report here that the minimum sequence requirement for the assembly of an active prolyl 4-hydroxylase α2β2 tetramer in insect cell coexpression experiments is fulfilled by the PDI domain constructb′a′ but that the sequential addition of the band a domains greatly increases the level of enzyme activity obtained. In the assembly of active prolyl 4-hydroxylase tetramers, the a and b domains of PDI, but notb′ and a′, can in part be substituted by the corresponding domains of ERp57, a PDI isoform that functions naturally in association with the lectins calnexin and calreticulin. Thea′ domain of PDI could not be substituted by the PDIa domain, suggesting that both b′ anda′ domains contain regions critical for prolyl 4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids that contain the b′ domain can bind the 14-amino acid peptide Δ-somatostatin, as measured by cross-linking; however, binding of the misfolded protein “scrambled” RNase required the addition of domains ab or a′ of PDI. The human prolyl 4-hydroxylase α subunit has at least two isoforms, α(I) and α(II), which form with the PDI polypeptide the (α(I))2β2 and (α(II))2β2 tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the α(II) subunit than the α(I) subunit.

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Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M010656200

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This work was supported by grants from the Health Sciences Council of the Academy of Finland, the Finnish Center of Excellence Program 2000–2005 Grant 44843, and European Union Contract BIO 4 CT 960436.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Present address: Biocenter Oulu and Department of Biochemistry, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland.