Eukaryotic Translation Elongation Factor 1A (eEF1A) Domain I from S. cerevisiae Is Required but Not Sufficient for Inter-Species Complementation

Sandra Eltschinger,Peter Bütikofer,Michael Altmann,Eva Greganova,Manfred Heller,Thomas Preiss

doi:10.1371/journal.pone.0042338

Abstract

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.

Highlights

Eukaryotic translation elongation factor 1A is a Gprotein which delivers aminoacyl-tRNAs to the ribosomal A-site and allows for proper codon-anticodon mediated deciphering of the genetic code
Our results using in vivo labeling and mass spectrometry demonstrate that Ethanolamine phosphoglycerol (EPG) is attached to E374 of HA-tagged HsEF1A expressed in T. brucei procyclic forms in culture
Since the enzymes involved in EPG attachment have not been characterized so far, we can only hypothesize if there is one phylogenetically ancient enzyme present in T. brucei, which later during evolution developed the capacity to modify domain II of Eukaryotic translation elongation factor 1A (eEF1A) in mammalian and plant cells, or if two different EPG modifying enzyme systems exists in these multicellular organisms

Summary

Introduction

Eukaryotic translation elongation factor 1A (eEF1A) is a Gprotein which delivers aminoacyl-tRNAs (aa-tRNAs) to the ribosomal A-site and allows for proper codon-anticodon mediated deciphering of the genetic code. This reaction requires hydrolysis of GTP to GDP and is assisted by eEF1B, a guanine nucleotide exchange factor that regenerates GTP-bound eEF1A and in S. cerevisiae consists of two subunits, eEF1Balpha which interacts directly with eEF1A and eEF1Bgamma. The primary structure of eEF1A is highly conserved among all eukaryotes and prokaryotes (for which it is termed EF-Tu). The crystal structure of yeast eEF1A has been elucidated, it consists of three domains termed I, II and III [2,3]. Whereas domain I binds guanine nucleotides, domains II and III are involved in the binding of aminoacyl-tRNAs [2] and eEF1Balpha binds in the hydrophobic pocket between domains I and II [3]

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