Several endoplasmic reticulum stress proteins, including ERp72, interact with thyroglobulin during its maturation.

We have previously demonstrated that several endoplasmic reticulum (ER) proteins, including BiP, ERp72, grp94, and protein disulfide isomerase, bind to a denatured thyroglobulin (Tg) affinity column and can be specifically eluted by ATP (Nigam, S.K., Goldberg, A.L., Ho, S., Rohde, M.F., Bush, K.T., and Sherman, M.Y. (1994) J. Biol. Chem. 269, 1744-1749). Using chemical cross-linking, we now demonstrate that BiP, ERp72, and grp94 associate with Tg in two types of cultured thyroid cells, FRTL-5 and PCC13. Whereas BiP could be coimmunoprecipitated with anti-Tg antibodies in the absence of cross-linking, only trace amounts of ERp72 and grp94 were coimmunoprecipitated. Likewise, in both cell types, anti-BiP antibodies were able to coimmunoprecipitate Tg in the absence of cross-linking, though ERp72 and grp94 were only minimally present. Coprecipitation of BiP and Tg was abolished when ATP and Mg2+ were added to cell lysates. In contrast, after cross-linking, there was a large increase in the amount of ERp72 and grp94 that coimmunoprecipitated with anti-Tg antibodies, although there was only a slight increase in BiP. Similarly, in cross-linked lysates, grp94 and ERp72 were also coimmunoprecipitated with anti-BiP antibodies. An apparently novel 200-kDa protein was also consistently immunoprecipitated by anti-BiP antibodies in both cell types. In addition, anti-ERp72 antibodies coimmunoprecipitated Tg, BiP, and grp94 only after cross-linking. Analysis of uncross-linked and cross-linked samples by sucrose density gradient centrifugation confirmed that Tg, BiP, grp94, and ERp72 are present together in high molecular weight complexes only after treatment of cells with cross-linking reagent. These results suggest that ERp72, as well as BiP and grp94, function as molecular chaperones in the maturation of Tg, potentially as part of a macromolecular complex.