Thyroglobulin Transport along the Secretory Pathway

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.

Thyroglobulin Is Selected as Luminal Protein Cargo for Apical Transport via Detergent-resistant Membranes in Epithelial Cells

“…By contrast, the pool of endoglycosidase H-resistant intracellular Tg, even at late chase times, was quite small (Fig. 1B), Thus, in MDCK cells as in CHO cells (36,40), recombinant Tg transport from Golgi to surface is a relatively rapid step so that the cells maintain only a small intracellular Golgi/post-Golgi pool of Tg at any moment.…”

“…Materials-The cDNA construct expressing bovine Tg was described previously (36). Rabbit polyclonal antibodies to Tg and to caveolin were obtained from Dr. P. R. Larsen (Brigham and Women's Hospital, Boston, MA) and Transduction Laboratories (Lexington, KY), respectively.…”

“…5) rendered unlikely the possibility that the protein failed to undergo ER-to-Golgi transport. Moreover, unlike CHO cells (36,40), COS cells (46), or MDCK cells (see above), as measured by immunofluorescence microscopy of permeabilized FRT cells, the predominant pool of recombinant Tg in the steady state was not in the ER but exhibited primarily a perinuclear pattern (Fig. 6C).…”

“…Fraction-Because of its large size, a full-length expressible Tg cDNA has become available only relatively recently (36), and the secretory polarity of the recombinant protein has never been examined. We therefore prepared stably transfected MDCK cells expressing Tg and selected positively expressing clones based on immunoblotting of conditioned media.…”

Cell Type-specific Storage of Dopamine β-Monooxygenase

“…This is in sharp contrast to the effect of expression of syntaxin 1A on the Golgi complex and endoplasmic reticulum of cells that do not normally express it (81); co-expression with rbSec1 allows syntaxin 1A to localize to the plasma membrane. Similarly, accessory proteins that bind to cellubrevin in rat liver (82), Factor VIII in AtT-20 cells (83), and thyroglobulin in CHO cells (84) have been identified. Further studies will be required to determine the mechanism underlying the cell type-specific trafficking of DBM to secretory granules.…”