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.
Highlights
Secretory proteins are translocated into the lumen of the endoplasmic reticulum (ER)1 in an unfolded form that is biologically inactive and incompetent for intracellular transport
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
Compared with CHO-P cells, there was no appreciable change in the total amount of rough ER in CHO-G cells, based on the levels of ribophorin II and calnexin (Fig. 2C), which are among the membrane proteins that appear to reflect the cellular quantity of rough ER [41, 42]
Summary
INVESTIGATION OF THE ROLE OF MOLECULAR CHAPERONE, GRP94, IN PROTEIN EXPORT FROM THE ENDOPLASMIC RETICULUM*. 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. We have been interested to know more about the effects of binding of GRP94 on the folding and export of thyroglobulin (Tg), the secretory protein precursor for thyroid hormone synthesis, and one of the reported “substrates” for GRP94 association in thyroid epithelial cells [18, 26, 27].
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