Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1α reveals a new post-translational protein modification

T. L. Rosenberry, J. A. Krall, T. E. Dever, R. Haas, D. Louvard, W. C. Merrick

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Abstract

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E.J., and Tartakoff, A.M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1α revealed that the protein was EF-1α. Copurification of rabbit EF-1α and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1α protein sequence deduced from a human EF-1α cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.

Original languageEnglish (US)
Pages (from-to)7096-7099
Number of pages4
JournalJournal of Biological Chemistry
Volume264
Issue number13
StatePublished - Jan 1 1989

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Rosenberry, T. L., Krall, J. A., Dever, T. E., Haas, R., Louvard, D., & Merrick, W. C. (1989). Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1α reveals a new post-translational protein modification. Journal of Biological Chemistry, 264(13), 7096-7099.