Folding and assembly of newly synthesized thyroglobulin occurs in a pre-Golgi compartment.
We have investigated the kinetics of folding and dimerization of newly synthesized thyroglobulin (Tg), the precursor protein in the manufacture of thyroid hormone. From the examination of lysates of pulse-labeled cultured thyrocytes by denaturing and nondenaturing gel electrophoresis, we have found that the earliest detectable form of nascent Tg is a transient aggregate, whose dissolution in vitro requires the addition of a reducing agent. In vivo, aggregate dissolution occurs with a t1/2 approximately 10 min at 37 degrees C. By 10 min after synthesis, monomeric Tg is first detectable in a conformationally unstable form. Dimeric Tg is formed thereafter (t1/2 approximately 30 min), but well before arrival of the protein in the medial Golgi (t1/2 approximately 2 h). Certain metabolic inhibitors permit dimerization yet block transport of the dimer to the Golgi. Thus, Tg dimerization occurs in a pre-Golgi compartment, and other steps after dimerization are likely to be important in the process of exit from the endoplasmic reticulum (ER). Further, aggregate dissolution, as well as dimerization, are inhibited significantly at 15 degrees C, indicating thermal sensitivity of Tg folding over and above effects on vesicular transport. Inhibitors of Tg iodination have no effect on Tg dimerization or Golgi arrival. Pretreatment of thyrocytes with thyroid-stimulating hormone substantially accelerates Tg flux through the ER, by increasing the amount, as well as the rate, of Tg transport, possibly at the expense of a small fraction of Tg that appears refractory to dimerization. Inhibition of N-linked glycosylation by tunicamycin causes a complete block in intracellular Tg transport by inducing the formation of biologically irreversible aggregates, suggesting that glycosylation of Tg serves to prevent denaturation of the secretory protein within the ER lumen.
Kim
P S
PS
Division of Endocrinology, Beth Israel Hospital, Boston, Massachusetts.
Arvan
P
P
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D006596:Hexosaminidases; D007700:Kinetics; D011233:Precipitin Tests; D011487:Protein Conformation; D013552:Swine; D013954:Thyroglobulin / Q000737:chemistry*; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D014415:Tunicamycin; D013954:Thyroglobulin; D006596:Hexosaminidases