Thyroglobulin Transport along the Secretory Pathway
INVESTIGATION OF THE ROLE OF MOLECULAR CHAPERONE, GRP94, IN PROTEIN EXPORT FROM THE ENDOPLASMIC RETICULUM*
- From the Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02215
Abstract
GRP94 serves as a molecular chaperone in the endoplasmic reticulum (ER). In normal thyrocytes, GRP94 interacts transiently with thyroglobulin (Tg), and in thyrocytes of animals suffering from congenital hypothyroid goiter with defective thyroglobulin, GRP94 and thyroglobulin associate in a protracted fashion. In order explore possible consequences of GRP94 binding, we have studied recombinant nonmutant thyroglobulin expressed in control Chinese hamster ovary (CHO) cells in comparison to that produced in CHO cells genetically manipulated for selectively increased GRP94 expression. Levels of ER chaperones other than GRP94 did not detectably differ, and thyroglobulin achieved transport competence in both kinds of CHO cells. However, increased availability of GRP94 caused the residence time of Tg in the ER to be remarkably prolonged. This was accompanied by a major increase in Tg directly associated with GRP94 and an increase in the ER pool size of Tg. Importantly, co-immunoprecipitation analysis revealed disulfide-linked Tg complexes (previously reported as an early Tg-folding intermediate) especially associated with GRP94. Indeed, non-native Tg, GRP94, and a 78-kDa protein likely to be BiP, appeared in ternary complexes. Under these conditions, GRP94 association appears directly involved in prolongation of Tg folding and export, consistent with a role in quality control in the ER.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant DK40344 (to P. A.) and a National Research Service Award postdoctoral fellowship (to Z. M.).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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↵‡ To whom correspondence should be addressed: Division of Endocrinology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8685; Fax: 718-430-8557; E-mail:arvan{at}aecom.yu.edu.
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↵1 The abbreviations used are: ER, endoplasmic reticulum; Tg, thyroglobulin; CHO, Chinese hamster ovary; CHO-P, CHO parental cells; CHO-G, GRP94-overexpressing cells; DSP, dithiobis(succinimdyl propionate); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.
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↵2 Increased occupancy and consequent redistribution of the KDEL receptor can also be excluded as an explanation for the retarded Tg export phenotype in CHO-G cells, because even when redistribution of this receptor is induced by lysozyme-KDEL overexpression, there is no evidence for perturbed flow of exportable proteins through the anterograde transport pathway (66).
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↵3 Z. Muresan and P. Arvan, manuscript submitted for publication.
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- Received December 31, 1996.
- Revision received July 17, 1997.
