Mannosylation of Endogenous Proteins of Rough and Smooth Endoplasmic Reticulum and of Golgi Membranes

Abstract

Mannosylation of the proteins of microsomal and Golgi membranes was investigated both after incubation in vitro of the isolated subfractions with GDP-[¹⁴C]mannose and after injection of [³H]mannose into rats followed by separation of these subfractions. Mannosylation of endogenous and added exogenous dolichol phosphate and also of dolichol pyrophosphate-oligosaccharide occurs in all three fractions. It was essential to inhibit antagonistic enzymes during incubation and to centrifuge after incubation. The presence of detergent in the incubation mixture influences the incorporation pattern of the different fractions in very different ways. In a system in vitro predominantly membrane proteins and not secretory proteins are mannosylated. Trypsin treatment of intact vesicles removes components from the outer surface only; such treatment liberates about one third of the radioactive mannose associated with lipid, releases radioactivity from the protein acceptor to the same extent and causes some inactivation of the transferase activities. It appears that a part of the mannosyl transferase system in rough and smooth endoplasmic reticulum and in Golgi membranes is localized at the cytoplasmic side of these membranes. This activity is probably involved in the glycosylation of proteins localized at the cytoplasmic surface of the endoplasmic reticulum.