Background: Intracellular myo-inositol homeostasis involves both de novo synthesis and uptake of myo-inositol from the environment. Results: Down-regulation of the myo-inositol transporter in Trypanosoma brucei causes depletion of bulk inositol lipids, but not glycosylphosphatidylinositols, and leads to parasite death. Conclusion: De novo synthesis of myo-inositol is not sufficient to ensure bulk inositol lipid production. Significance: myo-Inositol metabolism in T. brucei is compartmentalized.
Covalent modifications of proteins often modulate their biological functions or change their subcellular location. Among the many known protein modifications, three are exceptional in that they only occur on single proteins: ethanolamine phosphoglycerol, diphthamide and hypusine. Remarkably, the corresponding proteins carrying these modifications, elongation factor 1A, elongation factor 2 and initiation factor 5A, are all involved in elongation steps of translation. For diphthamide and, in part, hypusine, functional essentiality has been demonstrated, whereas no functional role has been reported so far for ethanolamine phosphoglycerol. We review the biosynthesis, attachment and physiological roles of these unique protein modifications and discuss common and separate features of the target proteins, which represent essential proteins in all organisms.
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.
2 SYNOPSISThe African trypanosome, Trypanosoma brucei, has been used as a model to study the biosynthesis of glycosylphosphatidylinositol (GPI) 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 inositol-acylated acyl-lyso-phosphatidylinositol (lyso-(acyl)PI) species, remain unaltered before protein attachment. By using a combination of metabolic labelling, cell-free assays and complementary mass spectrometry analyses, we now show 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 [ 3 H]myristatelabelled lipids in combination with ESI-MS/MS and MALDI-QIT-TOF-MS 3 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 polysaccharidelinked GPI anchors in procyclic form trypanosomes.Keywords: Glycosylphosphatidylinositol, Trypanosome, Lipid Biosynthesis, Lipid Remodelling, Posttranslational Modification, Mass Spectrometry.Abbreviations used: GPI, glycosylphosphatidylinositol; ESI-MS, electrospray ionizationmass spectrometry; ESI-MS-CID-MS, collision induced dissociation and tandem mass spectrometry; CESP, congolense epimastigote-specific protein; PRS, protease resistant surface molecule; PI, phosphatidylinositol; GARP, glutamic acid repetitive protein; JBAM, Jack bean -mannosidase; PI-PLC, phosphatidylinositol-specific phospholipase C; GPI-PLD, GPI-specific phospholipase D; MALDI-QIT-TOF-MS, matrix-assisted laserdesorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry; PA, phosphatidic acid; TLC, thin layer chromatography. A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law. in the types of GPI anchors they express. In T. brucei bloodstream forms, the GPI glycan core structure of variant surface glycoproteins (VSG), consisting of ethanolamine-phosphate-6-mannose-1,2-mannose-1,6-mannose-1,4-glucosamine-1,6-myo-inositol-1-phospholipid, is substituted with a short galactose branch [4, 5]. In contrast, in the procyclic stage, the GPI core of the procyclins is decorated with the largest and most complex GPI carbohydrate structure known today, consisting of a series of sialylated, branched N-acetyllactosamine/Nlactobiose repeats [6][7][8]. Moreover, while the lipid moiety of T. brucei bloodstream form GPI anchors consists exclusively of dimyristoyl phosphatidylinositol (PI) [4, 9], that of procyclic trypanosomes is composed of ...
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.
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