Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus.

Abstract

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.

Activated Thyroglobulin Possesses a Transforming Growth Factor-β Activity

“…Acidic pH, like denaturing agents, may induce a conformational change of Tg. Furthermore, the acidic pH effect may be physiologically relevant since Tg encounters acidic intracellular compartments during its secretion, uptake, and recycling by thyroid cells (23). As shown in Fig.…”

“…Growth Inhibition of Rat Thyroid Cells by DSS-activated Tg-As described previously, Tg may encounter intracellular acidic compartments during internalization and recycling in thyroid cells (2,23). The thyroid cells are presumably the target cells for activated Tg.…”

Effects of Tamoxifen, Toremifene and Chloroquine on the Lysosomal Enzymes in Cultured Retinal Pigment Epithelial Cells

“…Chloroquine has been found to raise the pH of acidic compartments and to prevent the dissociation of lysosomal enzymes from their receptors. The resulting lack of free receptors can prevent the delivery of newly synthetized enzymes to their cellular destinations (Gonzalez Noriega et al 1980;Miquelis et al 1993). This can be the direct reason for the decrease in the enzyme activities.…”

Extracellularly occurring histone H1 mediates the binding of thyroglobulin to the cell surface of mouse macrophages.

“…The thyroid lectin recognizes Tg molecules exposing N -acetylglucosamin in the terminal position of the N -linked glycans of Tg (13). It is believed to recycle low-iodinated Tg molecules via the Golgi apparatus to the apical plasma membrane, where Tg becomes available for iodination by the thyroid peroxidase and is released into the lumen of thyroid follicles (14).…”

“…However, further experiments showed that the 46-kD Tg-binding protein differed from the cationdependent mannose-6-phosphate receptor (12). Another Tg-binding protein on thyroid epithelial cells is the so-called thyroid lectin (13), which is believed to mediate uptake of low-iodinated Tg molecules from the lumina of thyroid follicles (14). The thyroid lectin recognizes Tg molecules exposing N -acetylglucosamin in the terminal position of the N -linked glycans of Tg (13).…”