Ethanolamine phosphoglycerol attachment to eEF1A is not essential for normal growth of Trypanosoma brucei

Abstract

Eukaryotic elongation factor 1A (eEF1A) is the only protein modified by ethanolamine phosphoglycerol (EPG). In mammals and plants, EPG is attached to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote, Trypanosoma brucei, a single EPG moiety is attached to domain III. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but the role of this unique protein modification in cellular growth and eEF1A function has remained elusive. Here we report, for the first time in a eukaryotic cell, a model system to study potential roles of EPG. By down-regulation of EF1A expression and subsequent complementation of eEF1A function using conditionally expressed exogenous eEF1A (mutant) proteins, we show that eEF1A lacking EPG complements trypanosomes deficient in endogenous eEF1A, demonstrating that EPG attachment is not essential for normal growth of T. brucei in culture.

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

“…The results showed that neither HsEF1A nor ScEF1A were able to complement the growth defect of TbEF1A-depleted T. brucei , whereas TbEF1A fully restored growth ( Fig. 2A , see also [20] ). Complementation of TbEF1A was further studied by conditionally expressing eEF1A from L. major (LmEF1A) in the C5 cell line, which is highly homologous to TbEF1A, differing in only 25 amino acids ( Fig.…”

“…Based on the above mentioned observations, we decided to investigate if HsEF1A and ScEF1A are able to complement T. brucei eEF1A (TbEF1A) function. In a previous study, we generated a tetracycline-inducible T. brucei cell line (named C5) in which expression of TbEF1A was ablated using RNAi, resulting in growth arrest of the parasites [20] . In the same work, we showed that growth could be fully restored by expressing an inducible ectopic copy of TbEF1A.…”

“…T. brucei procyclic form (PCF) Δprocyclin#1 (EP/GPEET null mutant) [18] and the derived cell lines were grown at 27°C in DTM supplemented with 15% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Basel, Switzerland) [19] . The T. brucei PCF “C5” RNAi cell line against T. brucei eEF1A [20] derived from strain 29-13 [21] was cultured at 27°C in SDM79 supplemented with 15% FBS and 25 μg/ml hygromycin, 15 μg/ml G-418 and 2 μg/ml puromycin. The T. brucei strain C5-E conditionally expressing an ectopic copy of T. brucei eEF1A [20] was cultured in the same medium with additional 10 μg ml −1 blasticidin S HCl (Invitrogen).…”

“…The T. brucei PCF “C5” RNAi cell line against T. brucei eEF1A [20] derived from strain 29-13 [21] was cultured at 27°C in SDM79 supplemented with 15% FBS and 25 μg/ml hygromycin, 15 μg/ml G-418 and 2 μg/ml puromycin. The T. brucei strain C5-E conditionally expressing an ectopic copy of T. brucei eEF1A [20] was cultured in the same medium with additional 10 μg ml −1 blasticidin S HCl (Invitrogen). Trypanosomes were stable transfected using 10–15 μg of linearized plasmid DNA and selected with 10 μg ml −1 blasticidin S HCl.…”

“…Afterwards, RNA was transferred from the gel to positively charged nylon membrane, Hybond-N + (GE Healthcare, Glattbrugg, Switzerland). Detection of polycistronic TbEF1A mRNA using a 32 P-labeled probe made by random priming of the PCR product of TbEF1A intergenic region 1, subsequent hybridization and analysis by autoradiography using Bio-Max MS film and a TransScreen-HE intensifying screen was performed as described previously [20] .…”