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 isomera se, bind to a denatured thyroglobulin (Tg) affinity column and can be specifically eluted by ATP (Nigam, S. K., Goldberg, A. L., Ho, S., Roh de, 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 c ells, FRTL-5 and PCCl3. Whereas BiP could be coimmunoprecipitated with anti-Tg antibodies in the absence of crosslinking, 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 p resent. Coprecipitation of BiP and Tg was abolished when ATP and Mg2were added to cell lysates. In contrast, after cross-linking, there wa s a large increase in the amount of ERp72 and grp94 that coimmunopreci pitated with anti-Tg antibodies, although there was only a slight incr ease in BiP. Similarly, in cross-linked lysates, grp94 and ERp72 were also coimmunoprecipitated with anti-BiP antibodies. An apparently nove l 200-kDa protein was also consistently immunoprecipitated by anti-BiP antibodies in both cell types. In addition, anti-ERp72 antibodies coi mmunoprecipitated Tg, BiP, and grp94 only after cross-linking. Analysi s of uncross-linked and cross-linked samples by sucrose density gradie nt centrifugation confirmed that Tg, BiP, grp94, and ERp72 are present together in high molecular weight complexes only after treatment of c ells with cross linking reagent. These results suggest that ERp72, as well as BiP and grp94, function as molecular chaperones in the maturat ion of Tg, potentially as part of a macromolecular complex.