Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1978 Jul;135(1):239–250. doi: 10.1128/jb.135.1.239-250.1978

Cell-free synthesis of proteins related to sn-glycerol-3-phosphate transport in Escherichia coli.

G Schumacher, K Bussmann
PMCID: PMC224812  PMID: 209011

Abstract

An Escherichia coli periplasmic protein (GlpT) related to sn-glycerol-3-phosphate transport was synthesized in a cell-free system directed by hybrid plasmic ColE1-glpT DNA. The in vitro product cross-reacted with antisera against the purified protein. The ColE1-glpT DNA-directed cell-free system was induced by sn-glycerol-3-phosphate and phosphonomycin and was dependent on cyclic AMP. The in vitro-synthesized protein showed the characteristics of a multimeric protein, as did the purified periplasmic protein. The main proportion of the newly synthesized product had a higher molecular weight than the mature protein found in the periplasm of cells and showed a more positive charge in two-dimensional gel electrophoresis. Thus, a proportion of this protein is presumed to be synthesized in vitro as a precursor. The cell-free system yielded a second protein that is likely to be also coded for by the glpT operon. This protein had a molecular weight of approximately 33,000 in sodium dodecyl sulfate-acrylamide gel electrophoresis and behaved like an intrinsic membrane protein.

Full text

PDF
239

Images in this article

242Image
on p.242
244Image
on p.244
245Image
on p.245
246Image
on p.246
247Image
on p.247
248Image
on p.248
249Image
on p.249

Selected References

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

  1. Argast M., Schumacher G., Boos W. Characterization of a periplasmic protein related to sn-glycerol-3-phosphate transport in escherichia coli. J Supramol Struct. 1977;6(1):135–153. doi: 10.1002/jss.400060111. [DOI] [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. Boos W., Hartig-Beecken I., Altendorf K. Purification and properties of a periplasmic protein related to sn-glycerol-3-phosphate transport in Escherichia coli. Eur J Biochem. 1977 Feb;72(3):571–581. doi: 10.1111/j.1432-1033.1977.tb11280.x. [DOI] [PubMed] [Google Scholar]
  4. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  5. Halegoua S., Hirashima A., Inouye M. Puromycin-resistant biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli. J Bacteriol. 1976 Apr;126(1):183–191. doi: 10.1128/jb.126.1.183-191.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Inouye H., Beckwith J. Synthesis and processing of an Escherichia coli alkaline phosphatase precursor in vitro. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1440–1444. doi: 10.1073/pnas.74.4.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Inouye S., Wang S., Sekizawa J., Halegoua S., Inouye M. Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1004–1008. doi: 10.1073/pnas.74.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johnson W. C., Silhavy T. J., Boos W. Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli. Appl Microbiol. 1975 Mar;29(3):405–413. doi: 10.1128/am.29.3.405-413.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Lin E. C. Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol. 1976;30:535–578. doi: 10.1146/annurev.mi.30.100176.002535. [DOI] [PubMed] [Google Scholar]
  11. Michaelis G., Saedler H., Venkov P., Starlinger P. Two insertions in the galactose operon having different sizes but homologous DNA sequences. Mol Gen Genet. 1969 Aug 15;104(4):371–377. doi: 10.1007/BF00334236. [DOI] [PubMed] [Google Scholar]
  12. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  13. Randall L. L., Hardy S. J. Synthesis of exported proteins by membrane-bound polysomes from Escherichia coli. Eur J Biochem. 1977 May 2;75(1):43–53. doi: 10.1111/j.1432-1033.1977.tb11502.x. [DOI] [PubMed] [Google Scholar]
  14. Schumacher G., Ehring R. RNA-directed cell-free synthesis of the galactose enzymes of Escherichia coli. Mol Gen Genet. 1973 Aug 28;124(4):329–344. doi: 10.1007/BF00267662. [DOI] [PubMed] [Google Scholar]
  15. Silhavy T. J., Hartig-Beecken I., Boos W. Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli. J Bacteriol. 1976 May;126(2):951–958. doi: 10.1128/jb.126.2.951-958.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Smith W. P., Tai P. C., Thompson R. C., Davis B. D. Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2830–2834. doi: 10.1073/pnas.74.7.2830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  18. Venkateswaran P. S., Wu H. C. Isolation and characterization of a phosphonomycin-resistant mutant of Escherichia coli K-12. J Bacteriol. 1972 Jun;110(3):935–944. doi: 10.1128/jb.110.3.935-944.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wetekam W., Staack K., Ehring R. DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli. Mol Gen Genet. 1971;112(1):14–27. doi: 10.1007/BF00266928. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES