The early stages in the biosynthesis of prothrombin

Prothrombin precursors undergo several modifications before secretion from the liver. These include signal-peptide cleavage (Friezner-Degen et al., 1983), complex glycosylation (Mizuochi et al., 1979) anddisulphide-bridge formation (Magnusson et al., 1975). Moreover, the function of plasma prothrombin is absolutely dependent upon the presence, at its N-terminus, of ten residues of y-carboxyglutamic acid (Stenflo et al., 1974). This novel amino acid is generated in the endoplasmic reticulum of the liver cell by the vitamin Kdependent carboxylation of specific glutamic acid residues in prothombin precursors (Olson & Suttie, 1977). At present, the temporal sequence and mechanism of the processing events responsible for the modification and secretion of prothrombin are poorly understood. We have employed a combination of approaches in oitro to define the steps in prothrombin biosynthesis. In order to locate the intracellular site of prothrombin synthesis, RNA was extracted (Palmiter, 1974) from endoplasmic reticulum-bound and cytosolic rat liver polysomes [fractionated according to Ikehara & Pitot (1973)l. When these RNAs were used to direct protein synthesis in a reticulocyte lysate (Pelham & Jackson, 1976), prothrombin was made only in response to the RNA from the membranebound polysomes. In a second series of experiments a prothrombinprocessing system was developed in vitro. Bovine prothrombin mRNA was partially purified by hybridization (Goldberg et al., 1979) to prothrombin complementary DNA (MacGillivray et al., 1980), cloned into the single-stranded fd phage vector fd 103 (Herrmann et al., 1980). When dog pancreas microsomes were added to a reticulocyte lysate