Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins*

Abstract

To determine the subcellular sites of fucose incorporation into TSH subunits, pituitaries from hypothyroid mice were incubated with [3H]fucose and fractionated by sucrose gradient centrifugation. To assess potential molecular cross-contamination between subcellular fractions enriched in rough endoplasmic reticulum (RER) or Golgi elements, trace amounts of exogenous [35S]methionine-labeled proteins or [125I]rat TSH were added before tissue homogenization. Particulate contamination of fractions was monitored by electron microscopy. TSH subunits were immunoprecipitated from fractions and analyzed by gel electrophoresis. After both a 2-h pulse incubation and a 2-h pulse, 3-h chase incubation, about half (range, 44-71%) of the [3H]fucose-labeled TSH subunit precursors present in microsomes were in the RER (amounts in excess of estimated contamination by nonspecific readsorption of molecules to vesicles or the presence of Golgi vesicles in the RER fractions); [3H]fucose-labeled free alpha-subunits were also detected in RER as well as in Golgi fractions. During chase incubations, both monensin and carboxyl cyanide m-chlorophenylhydrazone inhibited the appearance of [35S]methionine- or [3H]fucose-labeled TSH subunits in medium in a dose-dependent manner, suggesting that [3H] fucose was added to subunits, in part, early in the secretory pathway. Free alpha-subunits were more fucosylated than was TSH; in TSH heterodimers, beta-subunits were richer in fucose than were alpha-subunits. Thus, the fucosylation of TSH and free alpha-subunits in pituitaries of hypothyroid mice appears to begin at an unusually early stage of intracellular transport and may represent an adaptation to special posttranslational processing requirements.