A structural domain mediates attachment of ethanolamine phosphoglycerol to eukaryotic elongation factor 1A in Trypanosoma brucei.
Ethanolamine phosphoglycerol (EPG) represents a protein modification that so far has only been found in eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is covalently attached to two conserved glutamate residues located in domains II and III of eEF1A. In contrast, Trypanosoma brucei eEF1A contains a single EPG attached to Glu362 in domain III. The sequence and/or structural requirements for covalent linkage of EPG to eEF1A have not been determined for any organism. Using a combination of biosynthetic labelling of parasites with tritiated ethanolamine and mass spectrometry analyses, we demonstrate that replacement of Glu362 in T. brucei eEF1A by site-directed mutagenesis prevents EPG attachment, whereas single or multiple amino acid substitutions around the attachment site are not critical. In addition, by expressing a series of eEF1A deletion mutants in T. brucei procyclic forms, we demonstrate that a peptide consisting of 80 amino acids of domain III of eEF1A is sufficient for EPG attachment to occur. Furthermore, EPG addition also occurs if domain III of eEF1A is fused to a soluble reporter protein. To our knowledge, this is the first report addressing amino acid sequence, or structure, requirements for EPG modification of eEF1A in any organism. Using T. brucei as a model organism, we show that amino acid substitutions around the modification site are not critical for EPG attachment and that a truncated version of domain III of eEF1A is sufficient to mediate EPG addition.
Greganova
Eva
E
Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
Heller
Manfred
M
Bütikofer
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D017353:Gene Deletion; D005822:Genetic Vectors; D005990:Glycerol / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000235:genetics*; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D020648:Peptide Elongation Factor 1; D010714:Phosphatidylethanolamines; D005990:Glycerol