Phosphatidylethanolamine Is the Precursor of the Ethanolamine Phosphoglycerol Moiety Bound to Eukaryotic Elongation Factor 1A
2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês
10.1074/jbc.m802430200
ISSN1083-351X
AutoresAita Signorell, Jennifer Jelk, Monika Rauch, Peter Bütikofer,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoIn addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A. In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A. Eukaryotic elongation factor 1A (eEF1A) 2The abbreviations used are: eEF1A, eukaryotic elongation factor 1A; EPG, ethanolamine-phosphoglycerol; Etn, ethanolamine; GPI, glycosylphosphatidylinositol; PE, phosphatidylethanolamine; RNAi, RNA interference; Etn-P, ethanolamine-phosphate; CDP-Etn, CDP-ethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; HA, hemagglutinin; CM, chloroform:methanol; CMW, chloroform:methanol:water; GPI-phospholipase D, GPI-specific phospholipase D; MALDI-Tof/Tof, matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry; LC-MS/MS, liquid chromatography tandem mass spectrometry. is a member of the G-protein family and represents an essential component during protein synthesis by binding aminoacyl-tRNAs in a GTP-dependent reaction to the acceptor site of ribosomes during peptide chain elongation (1Negrutskii B.S. El'skaya A.V. Prog. Nucleic Acids Res. Mol. Biol. 1998; 60: 47-78Crossref PubMed Scopus (189) Google Scholar, 2Merrick, W. C., and Nyborg, J. (2000) in Translational Control of Gene Expression (Sonnenberg, N., Hershey, J. W. B., and Methews, M. B., eds) pp. 89-125, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar, 3Browne G.J. Proud C.G. Eur. J. Biochem. 2002; 269: 5360-5368Crossref PubMed Scopus (392) Google Scholar). Crystal structures of eEF1A in complex with components of the nucleotide exchange factor eEF1B have recently been reported (4Andersen G.R. Valente L. Pedersen L. Kinzy T.G. Nyborg J. Nat. Struct. Biol. 2001; 8: 531-534Crossref PubMed Scopus (101) Google Scholar, 5Andersen G.R. Nissen P. Nyborg J. Trends Biochem. Sci. 2003; 28: 434-441Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Besides its role during protein synthesis, eEF1A is involved in other cellular processes. It has long been proposed that eEF1A associates with and modulates microtubules and actin filaments in several cell types (1Negrutskii B.S. El'skaya A.V. Prog. Nucleic Acids Res. Mol. Biol. 1998; 60: 47-78Crossref PubMed Scopus (189) Google Scholar, 6Condeelis J. Trends Biochem. Sci. 1995; 20: 169-170Abstract Full Text PDF PubMed Scopus (258) Google Scholar). However, only recently could it be demonstrated that the conventional role of yeast eEF1A in protein synthesis and its non-canonical role in cytoskeleton organization clearly are separate functions (7Gross S.R. Kinzy T.G. Nat. Struct. Mol. Biol. 2005; 12: 772-778Crossref PubMed Scopus (218) Google Scholar). Furthermore, it has been reported that eEF1A, at least in the protozoan parasite Trypanosoma brucei, has yet another role in mediating the specificity of mitochondrial tRNA import (8Bouzaidi-Tiali N. Aeby E. Charriere F. Pusnik M. Schneider A. EMBO J. 2007; 26: 4302-4312Crossref PubMed Scopus (57) Google Scholar). Almost 20 years ago, two groups independently showed that eEF1A from a human erythroleukemia cell line (9Rosenberry T.L. Krall J.A. Dever T.E. Haas R. Louvard D. Merrick W.C. J. Biol. Chem. 1989; 264: 7096-7099Abstract Full Text PDF PubMed Google Scholar) and a murine lymphocyte cell line (10Whiteheart S.W. Shenbagamurthi P. Chen L. Cotter R.J. Hart G.W. J. Biol. Chem. 1989; 264: 14334-14341Abstract Full Text PDF PubMed Google Scholar) is modified by ethanolamine-phosphoglycerol (EPG), which is covalently attached to two conserved glutamate residues in the polypeptide chain (Fig. 1). Subsequently, the same modification was found in plant (11Ransom W.D. Lao P.-C. Gage D.A. Boss W.F. Plant Physiol. 1998; 117: 949-960Crossref PubMed Scopus (25) Google Scholar), but not yeast (12Cavallius J. Zoll W. Chakraburtty K. Merrick W.C. Biochim. Biophys. Acta. 1993; 1163: 75-80Crossref PubMed Scopus (60) Google Scholar), eEF1A. The discovery of the EPG modification was prompted by the observation that a 49-kDa cytosolic protein was labeled after incubation of mammalian cells in culture with tritiated ethanolamine (Etn) (9Rosenberry T.L. Krall J.A. Dever T.E. Haas R. Louvard D. Merrick W.C. J. Biol. Chem. 1989; 264: 7096-7099Abstract Full Text PDF PubMed Google Scholar, 10Whiteheart S.W. Shenbagamurthi P. Chen L. Cotter R.J. Hart G.W. J. Biol. Chem. 1989; 264: 14334-14341Abstract Full Text PDF PubMed Google Scholar), an approach that was originally aimed at identifying glycosylphosphatidylinositol (GPI)-anchored proteins. Etn is a component of the GPI core structure consisting of ethanolamine-phosphate-6-mannose-α1,2-mannose-α1,6-mannose-α1,4-glucosamine-α1,6-myo-inositol-1-phospholipid (13Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar). The pathway of Etn incorporation into GPI-anchored proteins involves uptake into cells via choline/ethanolamine transporter, incorporation into phosphatidylethanolamine (PE) via common phospholipid biosynthetic pathways, transfer of the Etn moiety from PE onto a GPI precursor lipid, and attachment of the GPI to a polypeptide precursor in the endoplasmic reticulum (14Eisenhaber B. Maurer-Stroh S. Novatchkova M. Schneider G. Eisenhaber F. BioEssays. 2003; 25: 367-385Crossref PubMed Scopus (148) Google Scholar, 15Kinoshita T. Inoue N. Curr. Opin. Chem. Biol. 2000; 4: 632-638Crossref PubMed Scopus (170) Google Scholar, 16Orlean P. Menon A.K. J. Lipid Res. 2007; 48: 993-1011Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Incorporation of tritiated ethanolamine into protein has also been observed for another lipid-modified protein, microtubule-associated protein 1 light chain 3 (17Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1117) Google Scholar). This protein, together with its yeast homologue Atg8, is modified by PE, which renders these proteins membrane-bound (17Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1117) Google Scholar, 18Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1519) Google Scholar). The attachment of PE to a C-terminal glycine residue in Saccharomyces cerevisiae Atg8 occurs in a ubiquitin-like conjugation reaction (18Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1519) Google Scholar). Recently, PE modification of Atg8 was shown to be linked to Atg8 function in membrane tethering and hemifusion during autophagosome formation (19Nakatogawa H. Ichimura Y. Ohsumi Y. Cell. 2007; 130: 165-178Abstract Full Text Full Text PDF PubMed Scopus (881) Google Scholar). At present, no information is available on the biosynthesis of EPG and its attachment to eEF1A, nor on its role in eEF1A function. Possible pathways for EPG synthesis include (i) the sequential addition of individual EPG components to the glutamate side chains of eEF1A, (ii) the pre-assembly of EPG, followed by its transfer to eEF1A, or (iii) the attachment of a larger structure containing the EPG moiety to eEF1A, followed by modification reactions on the protein. We favor this last possibility and hypothesize that eEF1A is modified with the phospholipid PE, followed by removal of the hydrophobic acyl or alkyl chains by appropriate enzymes. As a model system to address these questions, we chose T. brucei procyclic culture forms, because they readily take up tritiated Etn as a potential label for the EPG modification and because mutant cell lines can easily be generated to express tagged proteins, or down-regulate the expression of endogenous proteins using RNA interference (RNAi). In addition, as in most eukaryotic cells, the postulated substrate of the EPG modification, PE, represents a major phospholipid class in T. brucei (20Dixon H. Williamson J. Comp. Biochem. Physiol. 1970; 33: 111-128Crossref PubMed Scopus (57) Google Scholar, 21Patnaik P.K. Field M.C. Menon A.K. Cross G.A.M. Yee M.C. Bütikofer P. Mol. Biochem. Parasitol. 1993; 58: 97-106Crossref PubMed Scopus (65) Google Scholar). In mammalian cells and yeast, PE biosynthesis has been studied in great detail (22Vance J.E. Prog. Nucleic Acids Res. Mol. Biol. 2003; 75: 69-111Crossref PubMed Scopus (101) Google Scholar, 23Birner R. Daum G. Int. Rev. Cytol. 2003; 225: 273-323Crossref PubMed Scopus (28) Google Scholar). PE can be synthesized from Etn by phosphorylation to ethanolamine phosphate (Etn-P), which is subsequently activated to CDP-ethanolamine (CDP-Etn), and transferred onto diradylglycerol to form PE. This reaction sequence involving a CDP-activated intermediate has originally been delineated by Kennedy and Weiss (24Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar) and is commonly referred to as the Kennedy pathway. A similar reaction sequence involving a CDP-activated intermediate is also responsible for the synthesis of phosphatidylcholine (PC) (24Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). The individual reactions for PE and PC synthesis by the Kennedy pathway have been localized to the cytosol (25Vance J.E. Vance D.E. Biochem. Cell Biol. 2004; 82: 113-128Crossref PubMed Scopus (267) Google Scholar). Alternatively, PE can be generated by decarboxylation of phosphatidylserine (PS) or by head group exchange with PS. PS decarboxylation represents the major route for PE synthesis in mammalian cells and occurs in the Golgi and mitochondria (22Vance J.E. Prog. Nucleic Acids Res. Mol. Biol. 2003; 75: 69-111Crossref PubMed Scopus (101) Google Scholar, 26Vance J.E. Steenbergen R. Prog. Lipid Res. 2005; 44: 207-234Crossref PubMed Scopus (363) Google Scholar, 27Choi J.Y. Wu W.I. Voelker D.R. Anal. Biochem. 2005; 347: 165-175Crossref PubMed Scopus (25) Google Scholar). In T. brucei, the pathways for the synthesis of PE, or other phospholipid classes, have not been studied in detail (28Vial H.J. Eldin P. Tielens A.G.M. van Hellemond J.J. Mol. Biochem. Parasitol. 2003; 126: 143-154Crossref PubMed Scopus (112) Google Scholar, 29van Hellemond J.J. Tielens A.G. FEBS Lett. 2006; 580: 5552-5558Crossref PubMed Scopus (28) Google Scholar). Based on our hypothesis that PE may be the precursor of the EPG modification of eEF1A, we studied if blocking PE biosynthesis in T. brucei procyclic (insect) forms using RNAi against enzymes of the Kennedy pathway affects attachment of EPG to eEF1A. Unless otherwise specified, all reagents were of analytical grade and were from Merck (Darmstadt, Germany), Sigma-Aldrich (Buchs, Switzerland) or MP Biomedicals (Tägerig, Switzerland). [1-3H]Ethan-1-ol-2-amine hydrochloride ([3H]Etn, 60 Ci mmol-1) and [9,10(n)-3H]myristic acid ([3H]myristate, 60 Ci mmol-1) were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). l-[3H(G)]Serine ([3H]serine, 29.5 Ci mmol-1) was from PerkinElmer Life Sciences. BioMax MS films were from GE Healthcare (Buckinghamshire, UK), and Kodak MBX films from Kodak SA (Lausanne, Switzerland). Trypanosomes and Culture Conditions—The T. brucei EP/GPEET procyclin null mutant Δprocyclin#1 (30Vassella E. Bütikofer P. Engstler M. Jelk J. Roditi I. Mol. Biol. Cell. 2003; 14: 1308-1318Crossref PubMed Google Scholar), and the derived procyclic cell line expressing tagged eEF1A, were cultured at 27 °C in DTM supplemented with 15% heat-inactivated fetal bovine serum (Invitrogen). T. brucei 29-13 procyclic forms (31Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1115) Google Scholar) (obtained from Paul Englund, John Hopkins University School of Medicine) were cultured at 27 °C in SDM-79 containing 15% heat-inactivated fetal bovine serum, 25 μg/ml hygromycin, and 15 μg/ml G418 to maintain constitutive expression of the T7 RNA polymerase and the tetracycline repressor. Derived RNAi strains, including the RNAi strain against T. brucei eEF1A (8Bouzaidi-Tiali N. Aeby E. Charriere F. Pusnik M. Schneider A. EMBO J. 2007; 26: 4302-4312Crossref PubMed Scopus (57) Google Scholar) (a kind gift of André Schneider, University of Fribourg), were cultured in the presence of an additional 2 μg/ml puromycin. The expression of double-stranded RNA was induced by the addition of 1 μg/ml tetracycline. T. brucei 427 bloodstream forms were cultured at 37 °C and 5% CO2 in HMI-9 containing 10% heat-inactivated fetal bovine serum. Differentiation of bloodstream forms to procyclic forms was induced by the addition of 6 mm cis-aconitate and transferring the cells to 27 °C in SDM-79 containing 15% heat-inactivated fetal bovine serum (32Bütikofer P. Vassella E. Ruepp S. Boschung M. Civenni M. Seebeck T. Hemphill A. Mookherjee N. Pearson T.W. Roditi I. J. Cell Sci. 1999; 112: 1785-1795Crossref PubMed Google Scholar). Protein synthesis was blocked in Δprocyclin#1 parasites by the addition of cycloheximide (50 μg/ml, final concentration) to the culture medium 30 min before the start of the experiment. Protein synthesis was measured by incorporation of [3H]serine into protein. Construction of Epitope-tagged T. brucei eEF1A—To express a hemagglutinin-tagged variant of eEF1A (HA-eEF1A), the annotated T. brucei eEF1A (TEF1) gene (GeneDB accession number Tb10.70.5670) was amplified by PCR with flanking HindIII and BamHI restriction sites (primers TbEF1A, supplemental Table S1), cloned into the TOP10F′ vector with a TOPO TA Cloning Kit (Invitrogen), and subsequently subcloned between HindIII and BamHI restriction sites into the pCorleone vector (a kind gift of Isabel Roditi, University of Bern) (33Ruepp S. Furger A. Kurath U. Kunz Renggli C. Hemphill A. Brun R. Roditi I. J. Cell Biol. 1997; 137: 1369-1379Crossref PubMed Scopus (112) Google Scholar). The vector was linearized with HindIII, and an oligonucleotide coding for the HA tag and flanked by two HindIII sites (primers HA, supplemental Table S1) was cloned into the same vector at the N terminus of the T. brucei eEF1A gene. Before transfection into Δprocyclin#1, the vector (pAShaEF) was linearized with NotI and SalI. RNAi-mediated Gene Silencing—Putative T. brucei ethanolamine kinase (GeneDB accession number Tb11.18.0017) and putative T. brucei ethanolamine-phosphate cytidylyltransferase (Tb11.01.5730) were down-regulated by RNAi-mediated gene silencing using stem loop constructs containing a puromycin resistance gene. Cloning the gene fragments into the vector pALC14 (a kind gift of André Schneider, University of Fribourg) was performed as described previously (34Bochud-Allemann N. Schneider A. J. Biol. Chem. 2002; 277: 32849-32854Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), using PCR products obtained with primers Tb0017 (spanning nucleotides 261–810 of Tb11.18.0017) and Tb5730 (spanning nucleotides 51–585 of Tb11.01.5730) (supplemental Table S1), resulting in plasmids pAS0017 and pAS5730, respectively. For transfection of T. brucei procyclic forms, the vectors were linearized with NotI. Stable Transfection of Trypanosomes—T. brucei procyclic forms were transfected with pAS0017, pAS5730, or pAShaEF and selected for antibiotic resistance by addition of 10 μg/ml blasticidin S HCl (Invitrogen) for Δprocyclin#1 cells or 2 μg/ml puromycin for 29-13 RNAi cells to the culture medium. Clones were obtained by limiting dilution. RNA Isolation and Northern Blot Analysis—Total RNA for Northern blotting was prepared by the standard acidic guanidium isothiocyanate method (35Chomczynski P. Sacchi N. Nat. Protoc. 2006; 1: 581-585Crossref PubMed Scopus (1401) Google Scholar). Total RNA (10 μg) was separated on formaldehyde agarose gels and transferred to GeneScreen Plus nylon membranes (PerkinElmer Life Sciences). The 32P-labeled probes were made by random priming of the same PCR products used as inserts in the stem-loop vector (Prime-a-Gene Labeling System, Promega, Madison, WI). Hybridization was performed overnight at 60 °C, and the membrane was analyzed by autoradiography using BioMax MS film and a TransScreen-HE intensifying screen (Kodak). Ribosomal RNA was visualized on the same gel by ethidium bromide staining to control for equal loading. Metabolic Labeling and Extractions—Trypanosomes were labeled with [3H]Etn or [3H]myristate for 2–20 h (36Bütikofer P. Ruepp S. Boschung M. Roditi I. Biochem. J. 1997; 326: 415-423Crossref PubMed Scopus (69) Google Scholar) and sequentially extracted with 2 × 10 ml of chloroform:methanol (CM, 2:1, by volume) to extract bulk phospholipids, followed by 3 × 5 ml chloroform:methanol:water (CMW, 10:10:3, by volume) to solubilize GPI precursors and free GPIs (30Vassella E. Bütikofer P. Engstler M. Jelk J. Roditi I. Mol. Biol. Cell. 2003; 14: 1308-1318Crossref PubMed Google Scholar, 37Field, M. C., and Menon, A. K. (1992) in Lipid Modification of Proteins: A Practical Approach (Hooper, N. M., and Turner, A. J., eds) pp. 155-190, IRL Press, OxfordGoogle Scholar). The resulting pellet was solubilized in 1% SDS. CMW fractions were pooled, dried under nitrogen, and partitioned between butan-1-ol (CMWbut) and water (CMWaqu) (37Field, M. C., and Menon, A. K. (1992) in Lipid Modification of Proteins: A Practical Approach (Hooper, N. M., and Turner, A. J., eds) pp. 155-190, IRL Press, OxfordGoogle Scholar). In some experiments, CMWbut extracts were treated with purified GPI-specific phospholipase D (GPI-phospholipase D) from bovine serum as described before (38Bütikofer P. Boschung M. Brodbeck U. Menon A.K. J. Biol. Chem. 1996; 271: 15533-15541Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). [3H]Etn-labeled acid-soluble metabolites were extracted from trypanosomes as described elsewhere (39Rifkin M.R. Strobos C.A.M. Fairlamb A.H. J. Biol. Chem. 1995; 270: 16160-16166Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). TLC—One-dimensional TLC was performed on Silica Gel 60 plates. To separate phospholipids, CM extracts were run in solvent system 1, composed of chloroform:methanol:acetic acid: water (25:15: 4:2, by volume). For separation of GPI precursors, CMWbut phases were run in solvent system 2, composed of chloroform:methanol:water (4:4:1, by volume). Ethanolamine metabolites were separated in solvent system 3, composed of 25% ammonium hydroxide:methanol:0.6% NaCl in water (1:10: 10, by volume). Radioactivity was detected by scanning the airdried plate with a radioisotope detector (Berthold Technologies, Regensdorf, Switzerland) and quantified using the Rita Star® software provided by the manufacturer. Alternatively, the plate was sprayed with En3hance (PerkinElmer Life Sciences) and exposed to MXB film at -70 °C. On all TLC plates, appropriate lipid standards were run alongside the samples. Lipid Phosphorous Determination—Phospholipid fractions were scraped from TLC plates and digested by boiling in perchloric acid, and the released inorganic phosphate was reacted with ammonium molybdate and quantified photometrically (40Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2879) Google Scholar). Each determination was accompanied by a series of inorganic phosphate standards. The assay was linear between 0 and 200 nmol of phosphate per tube. SDS-PAGE and Immunoblotting—Extracted proteins were separated on 12% polyacrylamide gels under reducing conditions (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). For detection of 3H-labeled proteins, gels were fixed, stained with Coomassie Brilliant Blue, soaked in Amplify (GE Healthcare), dried, and exposed to MBX films at -70 °C. For immunoblotting, proteins were transferred onto Immobilon P polyvinylidene difluoride membranes (Millipore, Bedford, MA) by semi-dry blotting. Mouse monoclonal antibody against eEF1A (α-EF, Upstate, Lake Placid, NY) was used at a dilution of 1:5000. Mouse monoclonal antibody against HA (α-HA, Covance, Berkeley, CA) was used at a dilution of 1:3000. Mouse monoclonal antibody against EP procyclin (α-EP), generously provided by Terry W. Pearson (University of Victoria), was used at a dilution of 1:2500. Primary antibodies were detected with secondary rabbit anti-mouse IgG conjugated to horseradish peroxidase (Dako, Baar, Switzerland) at a dilution of 1:5000 and using an enhanced chemiluminescence detection kit (Pierce). Immunoprecipitation—HA-eEF1A from trypanosomes lyzed in 0.1% Nonidet P-40 was immunoprecipitated with Anti-HA Affinity Matrix (Roche Applied Science) according to the manufacturer's instructions and boiled in sample loading buffer for SDS-PAGE. Mass Spectrometry—Immunoprecipitated HA-eEF1A was subjected to SDS-PAGE, followed by in-gel reductive alkylation and trypsin digestion. Identification of peptide masses was done at the FingerPrints Proteomics Facility, Wellcome Trust Biocenter, University of Dundee (Dundee, Scotland), using an ABI 4700 matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-Tof/Tof) mass spectrometer. The tryptic peptides were further analyzed by liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) using a Dionex Ultimate LC, equipped with a Pepmap C18 column (75 μm × 15 cm), coupled to an ABI Q-Trap 4000 mass spectrometer. The peptide-resolving part of the nano-LC gradient was from 1% to 40% acetonitrile in 0.1% formic acid over 20 min at 300 nl/min. Chemical Treatment of eEF1A—Immunoprecipitated [3H]Etn-labeled HA-eEF1A was incubated in 50 μl of 70% formic acid at 37 °C for 4 or 22 h. The reaction was stopped by diluting the sample with 150 μl of water. After drying in a SpeedVac, peptides were resuspended in sample loading buffer and analyzed by SDS-PAGE, followed by immunoblotting or fluorography. Alternatively, SpeedVac-dried SDS fractions of [3H]Etn-labeled Δprocyclin#1 parasites were incubated with 50 μl of 70% formic acid at 37 °C for 4 or 22 h, diluted with 150 μl of water, and analyzed as above. T. brucei eEF1A Is Labeled with [3H]Etn—Alignment of the deduced protein sequence of T. brucei eEF1A with carrot and mouse eEF1A shows 76% identity with both sequences (Fig. 2). In particular, the two glutamate residues shown to be modified with EPG in plant, Glu289 and Glu362 (11Ransom W.D. Lao P.-C. Gage D.A. Boss W.F. Plant Physiol. 1998; 117: 949-960Crossref PubMed Scopus (25) Google Scholar), and mammalian eEF1A, Glu301 and Glu374 (9Rosenberry T.L. Krall J.A. Dever T.E. Haas R. Louvard D. Merrick W.C. J. Biol. Chem. 1989; 264: 7096-7099Abstract Full Text PDF PubMed Google Scholar, 10Whiteheart S.W. Shenbagamurthi P. Chen L. Cotter R.J. Hart G.W. J. Biol. Chem. 1989; 264: 14334-14341Abstract Full Text PDF PubMed Google Scholar), are conserved in T. brucei eEF1A (Glu289 and Glu362). The original identification of EPG bound to eEFA1 in mammalian cells was prompted by the observation that eEF1A was labeled with [3H]Etn (9Rosenberry T.L. Krall J.A. Dever T.E. Haas R. Louvard D. Merrick W.C. J. Biol. Chem. 1989; 264: 7096-7099Abstract Full Text PDF PubMed Google Scholar, 10Whiteheart S.W. Shenbagamurthi P. Chen L. Cotter R.J. Hart G.W. J. Biol. Chem. 1989; 264: 14334-14341Abstract Full Text PDF PubMed Google Scholar). We used the same approach in T. brucei and found that incubation of procyclic forms lacking the genes for procyclins (Δprocyclin#1) with [3H]Etn resulted in labeling of a single strong band at 49 kDa (Fig. 3A, left panel). The reason to use this particular mutant cell line was to avoid possible misidentification of labeled bands with the multiple forms of procyclins, which readily incorporate large amounts of [3H]Etn into their GPI anchors (36Bütikofer P. Ruepp S. Boschung M. Roditi I. Biochem. J. 1997; 326: 415-423Crossref PubMed Scopus (69) Google Scholar, 42Field M.C. Menon A.K. Cross G.A.M. EMBO J. 1991; 10: 2731-2739Crossref PubMed Scopus (87) Google Scholar). In addition, we found that a band of similar molecular mass is also recognized by a monoclonal antibody against eEF1A (Fig. 3A, right panel). The band at 37 kDa likely represents a degradation product of eEF1A. Further evidence that the 49-kDa labeled protein is eEF1A was obtained from experiments using a T. brucei 29-13 procyclic cell line expressing an RNAi construct designed to knock down the expression of eEF1A (8Bouzaidi-Tiali N. Aeby E. Charriere F. Pusnik M. Schneider A. EMBO J. 2007; 26: 4302-4312Crossref PubMed Scopus (57) Google Scholar). The results show that, after induction of RNAi by the addition of tetracycline to the culture medium, the 49-kDa band is no longer labeled with [3H]Etn; in contrast, incorporation of label into GPI-anchored EP procyclin, which migrates as a broad band at ∼42 kDa, continued (Fig. 3B, left panel). Immunoblotting revealed that induction of RNAi against eEF1A resulted in complete disappearance of the 49- and 37-kDa bands that are recognized by eEF1A antibody in control cells (Fig. 3B, right panel). In addition, we found that when protein synthesis, as measured by incorporation of [3H]serine into total protein, is inhibited by the addition of cycloheximide to the culture medium (Fig. 3C, left panel), incorporation of label into eEF1A is blocked, indicating that the attachment of Etn to eEF1A occurs during, or shortly after, protein synthesis (Fig. 3C). Furthermore, the 49-kDa [3H]Etn-labeled band was also seen in T. brucei 427 bloodstream forms and during differentiation of bloodstream to procyclic forms in culture (Fig. 3D). The additional 3H-labeled band at 55 kDa in T. brucei 427 bloodstream forms represents the variant surface glycoprotein, which is replaced during differentiation to procyclic forms by the EP/GPEET procyclins (43Roditi I. Liniger M. Trends Microbiol. 2002; 10: 128-134Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Together, these results strongly indicate that the 49-kDa [3H]Etn-labeled band in T. brucei procyclic and bloodstream forms represents eEF1A.FIGURE 3Ethanolamine incorporation into T. brucei eEF1A. A, after incubation of the procyclic T. brucei strain Δprocyclin#1 with 2.5 μCi/ml [3H]Etn for 18 h, trypanosomes were washed to remove unincorporated label, delipidated with organic solvents, and sequentially extracted as described under "Experimental Procedures." Proteins in the SDS extract were analyzed by SDS-PAGE and fluorography (left panel, 1 × 108 cell equivalents) or immunoblotting using α-EF antibody (right panel, 2 × 105 cell equivalents). B, endogenous eEF1A in T. brucei 29-13 procyclic forms was down-regulated using RNAi by incubating the cells in the absence (-) or presence (+) of tetracycline (Tet). After 8 h of induction, 1.5 μCi/ml [3H]Etn was added, and incubation was continued for an additional 16 h. SDS extracts were prepared and analyzed as in A. The lanes contain 2 × 108 and 5 × 106 cell equivalents for fluorography (left panel) and immunoblotting (right panel), respectively. C, T. brucei Δprocyclin#1 cells were incubated in the absence (-) or presence (+) of cycloheximide (CHX, 50 μg/ml final concentration) for 30 min to inhibit protein synthesis. Subsequently,
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