Biosynthesis of Rice Seed e-Amylase" Proteolytic Processing and Glycosylation Precursor Polypeptides by Microsomes of

Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of a-amylase, a secretory protein subject to proteolytic processing (cleavage of the N-terminal signal sequence) as well as glycosylation during its biosynthesis. The characterization and identification of the immunoprecipitable products synthesized were performed by SDS gel electrophoresis and subsequent fluorography. The molecular weight of the t~-amylase synthesized by the microsomes was found to be identical with that of the mature secretory form of the enzyme on the basis of electrophoretic mobilities. A significant portion of the enzyme molecules synthesized was shown to be segregated into the microsomal vesicles and protected against digestion by endo-fl-N-acetylglucosaminidase, indicating that both proteolytic processing and glycosylation of the precursor polypeptide chains take place in the microsomes. The modification of the polypeptide chains was further examined by disrupting the microsomal membranes with Triton X-100. Detergent treatment of the microsomes prior to protein synthesis caused an inhibition of both proteolytic processing and glycosylation of the polypeptide chains, leading to the synthesis of the unprocessed nascent (precursor I), processed but nonglycosylated nascent (precursor I I) forms, in addition to the mature form of a-amylase. Furthermore, the results of time-sequence analysis of the inhibitory effect of Triton X-100 on the modification of the polypeptide chains have led us to conclude that both proteolytic processing and subsequent glycosylation occur in the microsomes during the biosynthesis of a-amylase. We have demonstrated that, in the early germinating stages of rice seeds, a-amylase molecules are synthesized in the scutellar epithelium and secreted into the endosperm tissues (1, 18). The a-amylase is a typical secretory protein, and it is known to be subject to glycosylation during its biosynthesis (l, 15). Ultrastructural studies have shown that, at the onset of germination, rough endoplasmic reticulum (RER) and Golgi bodies are prominent and well developed in the scutellar epithelium cells (19), indicating that these organelles are possibly engaged in the synthesis, intracellular transport, and secretion of the enzyme molecules. In recent years, the mechanism of biosynthesis of both secretory and membranous glycoproteins has been thoroughly studied in mammalian system. It is now well accepted that the NH2-terminal signal peptide is co-translationally cleaved after it enters the cisternal space of endoplasmic reticulum (ER) (4802 7). The proteolytic processing and glycosylation of the elongating polypeptide chain have been reported to be directed by the microsomal membranes in various experimental systems (21, 23). Although these two reactions are known to be closely coupled with chain elongation and the vectorial segregation of polypeptide chains (3, 11, 13, 20, 21, 23), the nature of their precise relationship has not been well characterized with any of the secretory or membrane glycoproteins. Previously, we have reported that the translation of poly(A)mRNA isolated from rice seed scuteUum results in the formation of the unprocessed nascent form of a-amylase (17). Subsequently, the formation of the processed nascent form of the enzyme was demonstrated by inhibiting the glycosylation of the precursor with tunicamycin (TM), a specific inhibitor of protein glycosylation via the dolichol pathway (15). The unprocessed nascent and processed nascent forms of a-amylase, ]HE JOURNAL OF CELL BIOLOGY • VOLUME 96 MARCH 1983 802-806 © The Rockefeller University Press • 0021-9525/83/03/0802/05 $1.00 on Jne 7, 2017 D ow nladed fom Published March 1, 1983