Abstract
Thyroid hormone synthesis by thyrocytes depends upon apical secretion of thyroglobulin (Tg), the glycoprotein prohormone. In stably transfected MDCK cells, recombinant Tg is also secreted apically. All secreted Tg has undergone Golgi carbohydrate modification, whereas most intracellular Tg (which is slow to exit the endoplasmic reticulum) is sensitive to digestion with endoglycosidase H. However, in MDCK cells and PC Cl3 thyrocytes, a subpopulation of newly synthesized recombinant and endogenous Tg, respectively, is recovered in a Triton X-100 insoluble, glycosphingolipid/cholesterol-enriched (GEM/raft) fraction, and this small subpopulation is overwhelmingly endoglycosidase H resistant. Upon apical secretion, Tg solubility is restored. Apical secretion of Tg is inhibited by cellular cholesterol depletion. In FRT cells, recombinant Tg becomes Triton X-100 insoluble within 60 min after synthesis and a portion is actually endoglycosidase H-sensitive, suggesting early Tg entry into GEMs/rafts. Interestingly in FRT cells, Tg remains associated with the apical plasma membrane upon exocytosis, and all surface Tg is GEM/raft-associated. Thus, Tg is the first secretory protein demonstrated to enter Triton X-100 insoluble membranes en route to the apical surface of epithelial cells. The data imply that Tg utilizes a cargo-selective mechanism for apical sorting.
Highlights
The functional unit of the thyroid gland is the thyroid follicle, each follicle representing a simple epithelium in which a monolayer of thyroid epithelial cells surrounds a central lumen [1]
Triton X-100-insoluble glycosphingolipid/cholesterol-enriched membranes (GEMs)/rafts have never yet been shown to carry any apical secretory proteins. Both gp80/clusterin and a truncated, soluble form of placental alkaline phosphatase have both been investigated and not recovered in conventional Triton X-100-insoluble GEM/ raft fractions, such proteins might dissociate from components of GEM/raft microdomains upon permeabilization with detergent [20, 21]
Apical targeting of Tg to the lumen of thyroid follicles is essential for thyroid hormone synthesis [2]
Summary
The functional unit of the thyroid gland is the thyroid follicle, each follicle representing a simple epithelium in which a monolayer of thyroid epithelial cells surrounds a central (apical) lumen [1]. In MDCK cells and PC Cl3 thyrocytes, a subpopulation of newly synthesized recombinant and endogenous Tg, respectively, is recovered in a Triton X-100 insoluble, glycosphingolipid/cholesterol-enriched (GEM/raft) fraction, and this small subpopulation is overwhelmingly endoglycosidase H resistant.
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