(A) The predicted three-dimensional structures of eEF1A from T. brucei (left), H. sapiens (middle) and the X-ray structure of S. cerevisiae (right) are illustrated to document structural similarities. The positions of the glutamate residues representing potential EPG modification sites are indicated. The nomenclature of domains I, II, III is indicated for S. cerevisiae eEF1A. (B) T. brucei Δprocyclin#1 expressing HA-tagged human (HA-HsEF1A) or yeast (HA-ScEF1A) eEF1A were incubated in the presence of [³H]Etn for 18 h. Proteins in cell lysates (L) and in supernatants (SN), wash solutions (W) and the final pellet after immunoprecipitation using anti-HA antibody (IP) were separated by SDS-PAGE and analyzed by immunoblotting using α-HA monoclonal antibody (α-HA; upper panels) or fluorography (lower panels). Lanes contain extracts from 1×10⁷ (for L, SN and W) or 1.8×10⁸ cell equivalents (for IP). Molecular mass markers (kDA) are indicated.

(A) T. brucei RNAi parasites expressing ectopic copies of TbEF1A, HsEF1A, ScEF1A or LmEF1A were cultivated in the absence (−) or presence (+) of tetracycline (tet) for 7 days. Each day, cultures were diluted to a cell density of 3×10⁶ cells/ml and incubated with fresh medium. Non-induced HsEF1A, ScEF1A and LmEF1A cell lines showed the same growth curve as non-induced cell line TbEF1A: for simplicity, only the growth curve for TbEF1A is shown (see also [20]). (B) Northern blots of total RNA extracted from parasites after 3 days of incubation in the absence (−) or presence (+) of tetracycline (tet) and hybridized with ³²P-labeled probes against the intergenic region 1 of T. brucei eEF1A (top); rRNA was used as a loading control (bottom). (C) RT-PCR analysis of eEF1A transcripts. cDNA was synthesized from transcripts of T. brucei RNAi parasites cultured in the absence (−) or presence (+) of tetracycline for 72 h using primers specific for the different eEF1A orthologs (Table S1b). Lanes containing cDNA or total RNA (negative controls) are indicated. (D) Complementation assays in S. cerevisiae strain TKY102 expressing as unique source endogenous eEF1A from a URA3-plasmid. Cells were transformed with plasmids carrying genes encoding for different eEF1A orthologs. Upon transformation (upper panel), cells were incubated for several days on a plate containing 5-fluoroorotic acid (5-FOA) which is toxic in the presence of the URA3 plasmid. Only transformants which were able to loose due to mitotic segregation the URA3-plasmid grew on 5-FOA containing medium (lower panel). The numbers represent wild-type ScEF1A (1), HA-TbEF1A (2), TbEF1A (3), LmEF1A (4), HsEF1A (5), vector pRS314 (6, negative control), CaEF1A (7), His_6x-ScEF1A (8), and ScEF1A-His_6x (9).

(A) Sequence alignment of conserved amino acid motifs separating domains I and II (upper panel) and II and III (lower panel) of eEF1A from different sources. To generate chimeric constructs, synthetic SpeI (triangle) or BamHI (arrow) cloning sites were introduced. (B) Schematic representation of chimeric constructs. Arrows and triangles indicate the positions of the cloning sites which were removed by site-directed mutagenesis to reconstruct the original eEF1A sequences. Numbers in brackets correspond to clones shown in Fig. 3C. (C) Complementation of S. cerevisiae strain TKY102 with chimeric eEF1A constructs. Upper panels: Yeast cells growing on plates after transformation with different chimeric constructs. Middle and bottom panels: Counterselection for the loss of endogenous eEF1A on plates containing 5-FOA at 25 or 30°C. Left panels: Complementation assays with chimeric constructs carrying cloning sites causing a N329K mutation in the case of the artificial BamHI-site (separating domains II and III) or I254T/G255S mutations in the case of the artificial SpeI-site (separating domains I and II). Right panels: complementation assays with chimeric constructs after reconstructing wild type eEF1A sequence motifs. (D) Growth properties of S. cerevisiae complemented with chimeric eEF1A constructs. (1) positive control with yeast eEF1A; (2) Chimeric yeast constructs carrying human domain III - without or with N329K mutation; (4) Chimeric yeast constructs carrying T. brucei domain III - without or with N329K mutation; (8) Chimeric yeast eEF1A construct carrying humain domain II.