Thyroid-stimulating hormone subunit processing and combination in microsomal subfractions of mouse pituitary tumor

Abstract

Mouse pituitary thyrotropic tumor minces were labeled with [35S]methionine and fractionated into rough microsomes, intermediate, and low density smooth microsomes. Thyroid-stimulating hormone subunits were mainly in rough microsomes after a 10-min pulse, but with increasing chase times the proportion in smooth microsomes increased. In rough microsomes, small amounts of an alpha subunit precursor of Mr = 11,000 and larger amounts of an alpha form of Mr = 18,000 were rapidly processed to a form of Mr = 21,000, while small amounts of a beta-subunit precursor of M r = 11,000 were processed to a form of Mr = 18,000. Most of the Mr = 18,000 and Mr = 21,000 subunit forms were converted by endoglycosidase H to forms of Mr = 11,000 to 12,000. Small amounts of endoglycosidase H-resistant forms appeared in low density smooth microsomes after a 30-min chase. Subunit combination was not detected at 10 min; combination was first detected at 20 min and increased progressively to a maximum of 61% of beta in the low density smooth microsomes at 60 min of chase. Although alpha of Mr = 11,000 and 18,000, and beta of Mr - 11,000, were not detected in thyroid-stimulating hormone, both endoglycosidase H-sensitive and -resistant alpha subunit of Mr = 21,000 and beta subunit of Mr = 18,000 were found combined. Thus, the rough endoplasmic reticulum (ER) contains only small amounts of nonglycosylated subunits (Mr = 11,000). The major subunit precursors contain one high mannose oligosaccharide (Mr = 18,000), with a second unit being added onto alpha in the rough ER. Combination of alpha (Mr = 21,000) with beta (Mr = 18,000) begins in the rough ER but occurs predominantly in the smooth ER/Golgi. Oligosaccharides of both combined and uncombined subunits are processed from high mannose to complex forms predominantly in the smooth ER/Golgi.