The subcellular sites of sulfation of mouse thyrotropin and free alpha subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins.
To determine the subcellular sites of sulfation of thyrotropin (TSH) and free alpha-subunits, mouse thyrotropic tumor minces were incubated simultaneously with [3H]Met and [35S]SO4 for 1 or 3h, homogenized, and fractionated by discontinuous sucrose gradient ultracentrifugation. Dual-labeled TSH or free alpha-subunits were immunoprecipitated, and analyzed by SDS-gel electrophoresis. Endoglycosidase F released all [35S], but little [3H], from the dual-labeled species, indicating that [35S]SO4 was incorporated into oligosaccharides of TSH and free alpha-subunits. Both [35S]TSH and [35S] free alpha-subunits were predominantly in Golgi fractions at 1 and 3 h, but small amounts were also detected in fractions enriched in rough endoplasmic reticulum (RER). Similar distributions of [35S]SO4-labeled species were noted in cell fractions prepared from mouse pituitaries. Pituitaries from hypothyroid mice were incubated with [3H]Met and [35S]SO4 for 2 h, then chased for 4 or 16 h in the absence or presence of 2 uM monensin (Mon) or 10 uM carboxyl cyanide m-chlorophenylhydrazone (CCCP). At 4h, release into the medium of [3H]TSH was inhibited 59% and 86% by Mon and CCCP, respectively; release of [35S]TSH was inhibited 28% and 46%. At 4h, release of [3H]free alpha-subunits was inhibited 58% and 81% by these drugs, respectively; release of [35S]free alpha-subunits was inhibited 6% and 50%. Thus, Mon and CCCP inhibited the release of each [3H] species more than the [35S] species, indicating that most sulfation occurred in Golgi.
Magner
J A
JA
Department of Medicine, Michael Reese Hospital, University of Chicago, Illinois 60616.
Papagiannes
E
E
D000818:Animals; D001345:Autoradiography; D002458:Cell Fractionation / Q000379:methods; D006056:Golgi Apparatus / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D010902:Pituitary Gland / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism*
D011506:Proteins; D013431:Sulfates; D013972:Thyrotropin