Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

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. cerevisia

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

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

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

<i>In vivo</i> complementation assays in <i>T. brucei</i> and <i>S. cerevisiae</i> depleted for endogenous eEF1A.

<p>(<b>A</b>) <i>T. brucei</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042338#pone.0042338-Greganova3" target="_blank">[20]</a>). (<b>B</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 <i>T. brucei</i> eEF1A (top); rRNA was used as a loading control (bottom). (<b>C</b>) RT-PCR analysis of eEF1A transcripts. cDNA was synthesized from transcripts of <i>T. brucei</i> RNAi parasites cultured in the absence (−) or presence (+) of tetracycline for 72 h using primers specific for the different eEF1A orthologs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042338#pone.0042338.s002" target="_blank">Table S1b</a>). Lanes containing cDNA or total RNA (negative controls) are indicated. (<b>D</b>) Complementation assays in <i>S. cerevisiae</i> 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).</p

Lipid remodelling of glycosylphosphatidylinositol (GPI) glycoconjugates in procyclic-form trypanosomes: biosynthesis and processing of GPIs revisited

The African trypanosome, Trypanosoma brucei, has been used as a model to study the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. In mammalian (bloodstream)-form parasites, diacyl-type GPI precursors are remodelled in their lipid moieties before attachment to variant surface glycoproteins. In contrast, the GPI precursors of insect (procyclic)-form parasites, consisting of lyso-(acyl)PI (inositol-acylated acyl-lyso-phosphatidylinositol) species, remain unaltered before protein attachment. By using a combination of metabolic labelling, cell-free assays and complementary MS analyses, we show in the present study that GPI-anchored glycoconjugates in T. congolense procyclic forms initially receive tri-acylated GPI precursors, which are subsequently de-acylated either at the glycerol backbone or on the inositol ring. Chemical and enzymatic treatments of [3H]myristate-labelled lipids in combination with ESI-MS/MS (electrospray ionization-tandem MS) and MALDI-QIT-TOF-MS3 (matrix-assisted laser-desorption ionization-quadrupole ion trap-time-of-flight MS) analyses indicate that the structure of the lipid moieties of steady-state GPI lipids from T. congolense procyclic forms consist of a mixture of lyso-(acyl)PI, diacyl-PI and diacyl-(acyl)PI species. Interestingly, some of these species are myristoylated at the sn-2 position. To our knowledge, this is the first demonstration of lipid remodelling at the level of protein- or polysaccharide-linked GPI anchors in procyclic-form trypanosomes

Sapphires related to alkali basalts from the Cerova Highlands, Western Carpathians (southern Slovakia): composition and origin

Blue, grey-pink and pink sapphires from the Cerová Highlands, Western Carpathians (southern Slovakia) have been studied using CL, LA-ICP-MS, EMPA, and oxygen isotope methods. The sapphire occurs as (1) clastic heavy mineral in the secondary sandy filling of a Pliocene alkali basaltic maar at Hajnáčka, and (2) crystals in a pyroxenebearing syenite/anorthoclasite xenolith of Pleistocene alkali basalt near Gortva. Critical evaluation of compositional diagrams (Fe, Ti, Cr, Ga, Mg contents, Fe/Ti, Cr/Ga, Ga/Mg ratios) suggests a magmatic origin for clastic blue sapphires with lower Cr and Mg, but higher Fe and Ti concentrations in comparison to the grey-pink and pink varietes, as well as similar compositional trends with blue sapphire from the Gortva magmatic xenolith. Moreover, blue sapphires show similar δ&lt;sup&gt;18&lt;/sup&gt;O values: 5.1 ‰ in the Gortva xenolith, 3.8 and 5.85 ‰ in the Hajnáčka placer, closely comparable to mantle to lower crustal magmatic rocks. On the contrary, pink and grey-pink sapphires show higher Cr and Mg, but lower Fe and Ti contents and their composition points to a metamorphic (metasomatic) origin