Studies on the posttranslational migration and processing of thyroglobulin: use of inhibitors and evaluation of the role of phosphorylation.

Abstract

A study of the synthesis and migration of thyroglobulin has been carried out by the incubation of calf thyroid slices in the presence of [14C]leucine followed by the separation of intracellular compartments from the colloid by differential and density gradient centrifugation. The effect of inhibitors on these processes has been examined and, moreover, the importance of phosphorylation as an additional posttranslational modification of thyroglobulin has been explored through the characterization of the radiolabeled constituents formed after slice incubation with [32P]phosphate. Movement of newly synthesized thyroglobulin through the cellular compartments was found to be rapid; after a 15-min incubation with [14C]leucine a substantial portion (30%) of the immunoprecipitable radiolabeled thyroglobulin was already present in the colloid (soluble) fraction. Incubations performed after brief pretreatment with colchicine resulted in the accumulation of radiolabeled thyroglobulin in the colloid, suggesting that in the uninhibited state a large proportion of the newly synthesized thyroglobulin is reabsorbed and degraded. Vinblastine and cytochalasin B had similar but smaller effects. More prolonged preincubation with colchicine led to a substantial decrease in thyroglobulin formation, suggesting that the synthetic and reabsorptive steps have different degrees of sensitivity to agents affecting the microtubule-microfilament apparatus. Uptake of radiolabeled thyroglobulin from the colloid was also inhibited by chloroquine, which caused a block in the lysosomal degradation of this protein but did not affect its synthesis. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of immunoprecipitated thyroglobulin prepared after incubation of slices with radiolabeled substrates was that of a doublet. When, however, the incubation of either calf or rat thyroid segments was terminated by boiling in sodium dodecyl sulfate-2-mercaptoethanol, one band predominated, corresponding to the slowest of the immunoprecipitated components; it is suggested that the thyroglobulin molecule contains regions of great protease sensitivity which are responsible for the heterogeneous high mol wt pattern usually seen, but that physiologically this peptide trimming does not occur before secretion from the cell. Incubation of thyroid slices with [32P]phosphate led to incorporation of label into immunoprecipitable thyroglobulin associated with both the intracellular and colloid compartments.(ABSTRACT TRUNCATED AT 400 WORDS)