Incorporation of [3H]ethanolamine into a single cytosolic protein in a cell free system: ethanolaminylation of EF-1 alpha in vitro.
Ethanolamine containing modifications of cytosolic proteins have only been described for elongation factor 1 alpha (EF-1 alpha) which contains two ethanolamine-phosphoglycerol moieties at Glu 301 and 374 (Whiteheart et al. (1989) J. Biol. Chem. 264, 14334-14341 and Dever et al. (1989) J. Biol. Chem. 264, 20518-20525). In this report, we describe a cell-free, cytosolic extract which specifically incorporates [3H]ethanolamine into a single cytosolic protein with properties identical to EF-1 alpha. The incorporation reaction is dependent on time and extract and is independent of any membrane-bound components. The single in vitro-radiolabeled protein is modified on two sites and chromatographic analysis of pronase-digested peptides is consistent with the addition of an unmodified ethanolamine. Ethanolaminylation does not require divalent cations or ATP but is inhibited by N-ethylmaleimide and stimulated by reducing agents (beta-mercaptoethanol and dithiothreitol), indicating the requirement for free sulfhydryls. The nucleophile, hydroxylamine, at low concentrations, greatly inhibits the incorporation reaction, indicating the importance of an electrophilic center. This cytosolic extract appears to be able to carry out only the initial step in the addition of the ethanolamine-phosphoglycerol moieties to EF-1 alpha, and subsequent addition of the phosphoglycerol moiety appears to require membrane components.
Whiteheart
S W
SW
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Hart
G W
GW
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D014316:Tritium
D004983:Ethanolamines; D006898:Hydroxylamines; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D014316:Tritium; D019811:Hydroxylamine; D019856:Ethanolamine; D008623:Mercaptoethanol; D011402:Pronase; D005033:Ethylmaleimide; D004229:Dithiothreitol