INTRACELLULAR ROUTING OF GLCNAC-BEARING MOLECULES IN THYROCYTES - SELECTIVE RECYCLING THROUGH THE GOLGI-APPARATUS

Previous experiments led us to speculate that thyrocytes contain a rec ycling system for GlcNAc-bearing immature thyroglobulin molecules whic h 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 int ernalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using I -125-GlcNAc-BSA and I-131-Man-BSA. We observed that the apparent intak e 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 (ch loroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = ap proximately 30 min) than the Man-BSA(t1/2 = -8 h). Subcellular homing was first studied by cell fractionation after internalization using I- 125-ovomucoid and I-131-BSA. During Percoll density gradient fractiona tion, endogenous thyroperoxidase was used to separate subsets of organ elles involved in the biosynthetic exocytotic pathway. Incubation of t he cell homogenate in the presence of DAB and H2O2 before cell fractio nation 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 eve ry time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also obser ved that: (a) ovomucoid sequestered in the Golgi-rich subfraction inco rporated [H-3]galactose; and (b) that part of internalized ovomucoid w as localized on the Golgi stacks as well as elements of the trans-Golg i, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on sol uble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.