Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Mar 1;96(3):802–806. doi: 10.1083/jcb.96.3.802

Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes

PMCID: PMC2112409  PMID: 6187753

Abstract

Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of alpha- 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 alpha-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-beta-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 II) forms, in addition to the mature form of alpha-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 alpha- amylase.

Full Text

The Full Text of this article is available as a PDF (747.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bielinska M., Boime I. mRNA-dependent synthesis of a glycosylated subunit of human chorionic gonadotropin in cell-free extracts derived from ascites tumor cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1768–1772. doi: 10.1073/pnas.75.4.1768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chua N. H., Schmidt G. W. Transport of proteins into mitochondria and chloroplasts. J Cell Biol. 1979 Jun;81(3):461–483. doi: 10.1083/jcb.81.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Glabe C. G., Hanover J. A., Lennarz W. J. Glycosylation of ovalbumin nascent chains. The spatial relationship between translation and glycosylation. J Biol Chem. 1980 Oct 10;255(19):9236–9242. [PubMed] [Google Scholar]
  6. Haugen T. H., Heath E. C. De novo biosynthesis of an enzymatically active precursor form of bovine pancreatic RNase. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2689–2693. doi: 10.1073/pnas.76.6.2689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Joseph T., Higgins V., Spencer D. Precursor Forms of Pea Vicilin Subunits: MODIFICATION BY MICROSOMAL MEMBRANES DURING CELL-FREE TRANSLATION. Plant Physiol. 1981 Feb;67(2):205–211. doi: 10.1104/pp.67.2.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Katz F. N., Lodish H. F. Transmembrane biogenesis of the vesicular stomatitis virus glycoprotein. J Cell Biol. 1979 Feb;80(2):416–426. doi: 10.1083/jcb.80.2.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kiely M. L., McKnight G. S., Schimke R. T. Studies on the attachment of carbohydrate to ovalbumin nascent chains in hen oviduct. J Biol Chem. 1976 Sep 25;251(18):5490–5495. [PubMed] [Google Scholar]
  10. Lingappa V. R., Lingappa J. R., Prasad R., Ebner K. E., Blobel G. Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein. Proc Natl Acad Sci U S A. 1978 May;75(5):2338–2342. doi: 10.1073/pnas.75.5.2338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miyata S., Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation. Plant Physiol. 1982 Jul;70(1):147–153. doi: 10.1104/pp.70.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Miyata S., Okamoto K., Watanabe A., Akazawa T. Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 10. IN VIVO AND IN VITRO SYNTHESIS OF alpha-AMYLASE IN RICE SEED SCUTELLUM. Plant Physiol. 1981 Dec;68(6):1314–1318. doi: 10.1104/pp.68.6.1314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Okamoto K., Murai T., Eguchi G., Okamoto M., Akazawa T. Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium. Plant Physiol. 1982 Sep;70(3):905–911. doi: 10.1104/pp.70.3.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  15. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sun S. M., Buchbinder B. U., Hall T. C. Cell-free Synthesis of the Major Storage Protein of the Bean, Phaseolus vulgaris L. Plant Physiol. 1975 Dec;56(6):780–785. doi: 10.1104/pp.56.6.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES