Processing to Endoglycosidase H-Resistant Thyrotropin Subunits Occurs in the Presence of Brefeldin-A: Evidence Favoring the Recycling of Golgi Membranes to the Rough Endoplasmic Reticulum in Mouse Thyrotrophs

Abstract

In order to assess the localization and physiologic redistribution of Golgi enzymes within mouse thyrotrophs, we studied the carbohydrate processing of TSH subunits in the presence of brefeldin A (BFA). Although this drug clearly causes endoglycosidase (endo) H-sensitive species to accumulate in most cell types, our purpose was to determine whether or not endoglycosidase H-resistant forms of free alpha-subunits and TSH subunits eventually accumulated in small but significant amounts within mouse thyrotrophic tumor cells or pituitary thyrotrophs incubated with BFA. This drug is known to block intracellular transport from the rough endoplasmic reticulum (RER) to the proximal Golgi. Stimulated thyrotrophs have been reported to have some Golgi enzymes active in their dilated RER. Accumulation of endo H-resistant forms in the presence of BFA might be explained by (1) drug-induced enhancement of Golgi to RER membrane recycling with further aberrant distribution of Golgi enzymes or (2) an uncharacteristic trapping of glycoproteins within Golgi elements that might be an unusual action of BFA peculiar to thyrotrophs. Free alpha-subunits and TSH were labeled in mouse thyrotrophic tumor tissue or pituitaries incubated in pulse-chase fashion with [35S]methionine in the absence or presence of BFA, carboxyl cyanide m-chlorophylhydrazone (CCCP), or swainsonine. The results in tumor and pituitary tissue were similar. In incubations without drugs, most TSH subunits (greater than 90%) became endo H-resistant after 5-h chase, and the majority (greater than 85%) were secreted. Doses of CCCP and BFA were selected that generally blocked the secretion of TSH subunits by greater than 85% (in some cases greater than 99%), presumably because of accumulation of secretory proteins in the RER. Yet, in the presence of CCCP, 35% and 42% of intracellular free alpha-subunits and TSH subunits, respectively, became endo H-resistant at 5 h chase. Compared to control incubations, intracellular subunits tended to remain endo H-sensitive in the presence of BFA, yet, compared to CCCP incubations, BFA slightly enhanced the attainment of endo H-resistance by free alpha-subunits and TSH subunits to 55% and 52%, respectively. Pretreatment of tumor tissue with BFA allowed more endo H-resistant species to appear, even during coincubation with CCCP. These data suggest that Golgi enzymes cycle back to the dilated RER of active thyrotrophs and that this phenomenon is enhanced by BFA.