Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1983 Apr 15;212(1):161–165. doi: 10.1042/bj2120161

Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes.

E M Danielsen, O Norén, H Sjöström
PMCID: PMC1152024  PMID: 6135417

Abstract

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.

Full text

PDF
161

Images in this article

162Fig. 1.
on p.162
163Fig. 2.
on p.163
164Fig. 3.
on p.164
164Fig. 4.
on p.164

Selected References

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

  1. Bar-Nun S., Kreibich G., Adesnik M., Alterman L., Negishi M., Sabatini D. D. Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 1980 Feb;77(2):965–969. doi: 10.1073/pnas.77.2.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braell W. A., Lodish H. F. Ovalbumin utilizes an NH2-terminal signal sequence. J Biol Chem. 1982 Apr 25;257(8):4578–4582. [PubMed] [Google Scholar]
  3. Braell W. A., Lodish H. F. The erythrocyte anion transport protein is contranslationally inserted into microsomes. Cell. 1982 Jan;28(1):23–31. doi: 10.1016/0092-8674(82)90371-3. [DOI] [PubMed] [Google Scholar]
  4. Browning T. H., Trier J. S. Organ culture of mucosal biopsies of human small intestine. J Clin Invest. 1969 Aug;48(8):1423–1432. doi: 10.1172/JCI106108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Danielsen E. M. Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase. Biochem J. 1982 Jun 15;204(3):639–645. doi: 10.1042/bj2040639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Danielsen E. M., Norén O., Sjöström H. Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide. Biochem J. 1982 Apr 15;204(1):323–327. doi: 10.1042/bj2040323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Danielsen E. M., Sjöström H., Norén O., Bro B., Dabelsteen E. Biosynthesis of intestinal microvillar proteins. Characterization of intestinal explants in organ culture and evidence for the existence of pro-forms of the microvillar enzymes. Biochem J. 1982 Mar 15;202(3):647–654. doi: 10.1042/bj2020647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Danielsen E. M., Skovbjerg H., Norén O., Sjöström H. Biosynthesis of intestinal microvillar proteins. Nature of precursor forms of microvillar enzymes from Ca2+-precipitated enterocyte membranes. FEBS Lett. 1981 Sep 28;132(2):197–200. doi: 10.1016/0014-5793(81)81159-3. [DOI] [PubMed] [Google Scholar]
  9. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kenny A. J., Maroux S. Topology of microvillar membrance hydrolases of kidney and intestine. Physiol Rev. 1982 Jan;62(1):91–128. doi: 10.1152/physrev.1982.62.1.91. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Snider M. D., Robbins P. W. Transmembrane organization of protein glycosylation. Mature oligosaccharide-lipid is located on the luminal side of microsomes from Chinese hamster ovary cells. J Biol Chem. 1982 Jun 25;257(12):6796–6801. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES