Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M802430200 on May 22, 2008

J. Biol. Chem., Vol. 283, Issue 29, 20320-20329, July 18, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/29/20320    most recent
M802430200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Signorell, A.
Right arrow Articles by Bütikofer, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Signorell, A.
Right arrow Articles by Bütikofer, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Phosphatidylethanolamine Is the Precursor of the Ethanolamine Phosphoglycerol Moiety Bound to Eukaryotic Elongation Factor 1A*Formula

Aita Signorell, Jennifer Jelk, Monika Rauch, and Peter Bütikofer1

From the Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, Bern 3012, Switzerland

Received for publication, March 28, 2008 , and in revised form, May 9, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic elongation factor 1A (eEF1A)2 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 (1–3). Crystal structures of eEF1A in complex with components of the nucleotide exchange factor eEF1B have recently been reported (4, 5). 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 (1, 6). 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 (7). 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 (8).

Almost 20 years ago, two groups independently showed that eEF1A from a human erythroleukemia cell line (9) and a murine lymphocyte cell line (10) 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 (11), but not yeast (12), 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) (9, 10), 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-{alpha}1,2-mannose-{alpha}1,6-mannose-{alpha}1,4-glucosamine-{alpha}1,6-myo-inositol-1-phospholipid (13). 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 (14–16).

Incorporation of tritiated ethanolamine into protein has also been observed for another lipid-modified protein, microtubule-associated protein 1 light chain 3 (17). This protein, together with its yeast homologue Atg8, is modified by PE, which renders these proteins membrane-bound (17, 18). The attachment of PE to a C-terminal glycine residue in Saccharomyces cerevisiae Atg8 occurs in a ubiquitin-like conjugation reaction (18). Recently, PE modification of Atg8 was shown to be linked to Atg8 function in membrane tethering and hemifusion during autophagosome formation (19).

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 (20, 21).


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 1.
Chemical structure of the EPG modification linked to the side chains of Glu301 and Glu374 of murine eEF1A (10).

 
In mammalian cells and yeast, PE biosynthesis has been studied in great detail (22, 23). 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 (24) 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) (24). The individual reactions for PE and PC synthesis by the Kennedy pathway have been localized to the cytosol (25). 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 (22, 26, 27). In T. brucei, the pathways for the synthesis of PE, or other phospholipid classes, have not been studied in detail (28, 29). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 {Delta}procyclin#1 (30), 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 (31) (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 (8) (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 (32).

Protein synthesis was blocked in {Delta}procyclin#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) (33). 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 {Delta}procyclin#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 (34), 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 {Delta}procyclin#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 (35). 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 (36) and sequentially extracted with 2 x 10 ml of chloroform:methanol (CM, 2:1, by volume) to extract bulk phospholipids, followed by 3 x 5 ml chloroform:methanol:water (CMW, 10:10:3, by volume) to solubilize GPI precursors and free GPIs (30, 37). 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) (37). In some experiments, CMWbut extracts were treated with purified GPI-specific phospholipase D (GPI-phospholipase D) from bovine serum as described before (38). [3H]Etn-labeled acid-soluble metabolites were extracted from trypanosomes as described elsewhere (39).

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 (40). 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 (41). 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 ({alpha}-EF, Upstate, Lake Placid, NY) was used at a dilution of 1:5000. Mouse monoclonal antibody against HA ({alpha}-HA, Covance, Berkeley, CA) was used at a dilution of 1:3000. Mouse monoclonal antibody against EP procyclin ({alpha}-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 x 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 {Delta}procyclin#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.


Figure 2
View larger version (78K):
[in this window]
[in a new window]

  		FIGURE 2.
Amino acid sequence comparison of T. brucei eEF1A with plant and mammalian eEF1A. The deduced amino acid sequence of the annotated T. brucei eEF1A gene (accession number Tb10.70.5670) was aligned with the deduced protein sequences of the corresponding genes from Daucus carota and Mus musculus using the ClustalW algorithm. White letters on the black background indicate amino acid identity between the sequences; black letters on the gray background indicate similarity. The EPG modification sites in D. carota (Ransom et al. (11)) and M. musculus (Rosenberry et al. (9) and Whiteheart et al. (10)) are marked with an asterisk.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (11), and mammalian eEF1A, Glu301 and Glu374 (9, 10), 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 (9, 10). We used the same approach in T. brucei and found that incubation of procyclic forms lacking the genes for procyclins ({Delta}procyclin#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 (36, 42). 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 (8). 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 (43). Together, these results strongly indicate that the 49-kDa [3H]Etn-labeled band in T. brucei procyclic and bloodstream forms represents eEF1A.


Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 3.
Ethanolamine incorporation into T. brucei eEF1A. A, after incubation of the procyclic T. brucei strain {Delta}procyclin#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 x 108 cell equivalents) or immunoblotting using {alpha}-EF antibody (right panel, 2 x 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 x 108 and 5 x 106 cell equivalents for fluorography (left panel) and immunoblotting (right panel), respectively. C, T. brucei {Delta}procyclin#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, trypanosomes were incubated with 2.5 µCi/ml [3H]Ser or 1.5 µCi/ml [3H]Etn for 8 h. Labeled proteins were extracted and analyzed as in A. The lanes contain 3 x 107 [3H]serine-labeled or 1 x 108 [3H]Etn-labeled cell equivalents for fluorography, and 5 x 106 cell equivalents for immunoblot analyses. D, T. brucei bloodstream forms were incubated at 37 °C with 0.3 µCi/ml [3H]Etn for 9 h. Labeled proteins were extracted and analyzed as in A (left lane). Differentiation of bloodstream forms to procyclic forms was triggered by the addition of 6 mM cis-aconitate to the culture medium and a temperature shift to 27 °C. After 0, 9, and 22 h of differentiation, trypanosomes were incubated an additional 9 h with 0.3 µCi/ml [3H]Etn. Labeled proteins were extracted and analyzed as above (right three lanes). The lanes contain 9 x 107 cell equivalents.

 
T. brucei eEF1A Is Modified with EPG at Glu362—To study the chemical nature of the Etn bound to T. brucei eEF1A, a HA-tagged form of eEF1A was transfected into T. brucei {Delta}procyclin#1 and isolated by immunoprecipitation using anti-HAcoated beads. The results show that isolated HA-tagged eEF1A is also labeled with [3H]Etn and is recognized by antibodies against eEF1A and HA (Fig. 4A). Analysis of immunoprecipitated HA-eEF1A by SDS-PAGE and Coomassie staining revealed two bands migrating very close to each other at 50 kDa (Fig. 4B). The bands were cut out individually and prepared for MS analysis by in-gel reduction and alkylation and digestion with trypsin. The resulting tryptic peptides were analyzed by MALDI-Tof/Tof MS. Both bands were thus identified as being T. brucei eEF1A, with sequence coverage of 50 and 42%, respectively. Both samples also exhibited ions at m/z 1020.465, consistent with being the [M+H]+ ions of the tryptic peptide FAEIESK (823.420 Da, amino acids 360–366 of the eEF1A sequence, see Fig. 2) plus a predicted mass increase of 197.045 Da resulting from the attachment of EPG to a glutamate residue. In contrast, the second tryptic peptide predicted to possibly contain an EPG modification at Glu289, SIEMHHEQLAEATPGDNVGFNVK (amino acids 279–301 of the eEF1A sequence, see Fig. 2), was only found in unmodified form, i.e. with [M+H]+ 2523.193 Da. At present, we don't know why HA-eEF1A runs as a doublet on SDS-PAGE.

To confirm that peptide FAEIESK was modified and to localize the modification, the same tryptic peptide samples were analyzed by nano-LC-MS/MS. In both samples, a peptide eluting at 13.3 min produced an [M+2H]2+ ion at m/z 510.8 that gave a product ion spectrum that clearly localized an EPG-modified glutamic acid residue (E*) in the sequence FAE*IESK. The spectrum is consistent with a facile elimination of glycerol phosphate, leaving an aminoethene modified Glu362 residue, as indicated in Fig. 5.

To further demonstrate that Glu362 is the only site modified with EPG, [3H]Etn-labeled eEF1A was treated with mild acid. Formic acid has been shown to selectively and efficiently cleave Asp–Pro bonds in proteins (44); in addition, it also cleaves Asp–Gly bonds, albeit at much lower efficiency (45). Because T. brucei eEF1A contains a single Asp–Pro bond located between the two potential EPG modification sites and only two Asp–Gly bonds, formic acid treatment was used to cleave [3H]Etn-labeled eEF1A into distinct peptides (Fig. 6A). Analysis by SDS-PAGE, followed by fluorography and immunoblotting, shows that treatment of endogenous or HA-tagged eEF1A with mild acid for 22 h generated a single labeled polypeptide of ~12 kDa (Fig. 6, B and D). Immunoblotting using {alpha}-EF shows that the 49-kDa band of eEF1A completely disappears during formic acid treatment and the band at ~37 kDa accumulates (Fig. 6C). Similarly, immunoblotting using {alpha}-HA shows the disappearance of the 50-kDa HA-eEF1A doublet and a transient accumulation of the 37-kDa band; after prolonged treatment with formic acid, the antibody detects a product of ~14 kDa (Fig. 6E). These results, in particular the absence of 3H-labeled fragments of 35 or 21 kDa, are consistent with the predicted cleavage pattern of eEF1A by formic acid (Fig. 6A) and demonstrate that T. brucei eEF1A contains a single EPG-modified site at Glu362 that is labeled with [3H]Etn.


Figure 4
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 4.
Characterization and isolation of HA-tagged eEF1A in T. brucei. T. brucei {Delta}procyclin#1 trypanosomes expressing an ectopic copy of HA-tagged T. brucei eEF1A were labeled with 1.5 µCi/ml [3H]Etn for 18 h. Subsequently, HA-eEF1A was isolated by immunoprecipitation and analyzed by SDS-PAGE and fluorography (A, top panel), or immunoblotting using {alpha}-EF or {alpha}-HA antibodies (A, bottom panels). The left lane of the fluorograph shows total 3H-labeled eEF1A in {Delta}procyclin#1 parasites (endogenous eEF1A plus HA-eEF1A), the right lane represents immunoprecipitated HA-eEF1A. The lanes contain 3.5 x 107 cell equivalents for fluorography and 5 x 106 cell equivalents for immunoblotting. In a separate experiment, HA-eEF1A was isolated from 8.5 x 107 non-labeled parasites by immunoprecipitation and analyzed by SDS-PAGE and Coomassie staining (B). The two bands at 50 kDa were subsequently excised and used for mass spectrometry analysis.

 


Figure 5
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 5.
Product ion spectrum of the EPG-modified peptide from T. brucei eEF1A. A tryptic digest of T. brucei eEF1A was subjected to nano-LC-MS/MS and an ion at m/z 510.8 was subjected to collision-induced dissociation to produce the spectrum in panel A. The spectrum can be assigned to conventional y- and b-type ions assuming that the EPG-modified glutamic acid residue (E*) undergoes rapid elimination of glycerol phosphate to generate an aminoethene-modified glutamic acid residue (Ex), as indicated in panel B, where the modified glutamic acid residue is indicated in italics. Other diagnostic ions are the y6 ion of the precursor peptide (FAE*IESK) at m/z 873.72 and the ethanolamine-phosphoglycerol ion at m/z 216.24.

 
Down-regulation of PE Biosynthesis—To test our working hypothesis that PE is the precursor of the EPG modification of eEF1A, we studied the effects of down-regulating possible PE biosynthetic pathways by RNAi on EPG attachment in T. brucei procyclic forms. Searching the T. brucei genome for homologues encoding enzymes involved in PE synthesis revealed candidate sequences for all three enzymes of the Kennedy pathway and, in addition, candidate sequences for PE/PS head group exchange and PS decarboxylation (supplemental Fig. S1). Subsequently, we targeted the first two putative enzymes of the Kennedy pathway, ethanolamine kinase (Tb11.18.0017) and ethanolamine-phosphate cytidylyltransferase (Tb11.01.5730), for RNAi using tetracycline-inducible expression of double-stranded RNA. The nucleotide sequences of the constructs used showed no significant homology to any other sequences in the trypanosome genome. Transfection of T. brucei procyclic forms with these constructs and selection by resistance to puromycin resulted in several mutant clones, two each of which were selected for all subsequent experiments.

Down-regulation of ethanolamine kinase or ethanolamine-phosphate cytidylyltransferase by RNAi resulted in a complete disappearance of the respective RNA (Fig. 7, top). After 3 days of culture in the presence of tetracycline, growth of the individual clones slowed down and, in the case of ethanolamine-phosphate cytidylyltransferase, ceased after 7 days of induction (Fig. 7, bottom). To study the effects of RNAi on PE biosynthesis, mutant clones were incubated in the presence of [3H]Etn and the incorporation of radioactivity into PE, or other lipids, was determined by TLC analysis. The results show that essentially all radioactivity in the lipid fraction co-migrated with a PE standard (Fig. 8A, left panels). After down-regulation of ethanolamine kinase or ethanolamine-phosphate cytidylyltransferase by RNAi for 3 days, the amount of labeled PE decreased to <30% of control levels (Fig. 8A, right panels). A block in PE synthesis at ethanolamine-phosphate cytidylyltransferase is expected to result in accumulation of the immediate biosynthetic precursor of the reaction, Etn-P. Thus, we analyzed the 3H-labeled acid-soluble metabolites and found that control trypanosomes show small amounts of radioactivity co-migrating with Etn-P and CDP-Etn standards, whereas RNAi cells after down-regulation of ethanolamine-phosphate cytidylyltransferase show an accumulation of [3H]Etn-P, and a lack of CDP-[3H]Etn (Fig. 8B).


Figure 6
View larger version (51K):
[in this window]
[in a new window]

  		FIGURE 6.
Analysis of EPG modification sites in T. brucei eEF1A. A, proposed cleavage pattern of T. brucei eEF1A during formic acid treatment. Endogenous (amino acids 1–449) or HA-tagged eEF1A (including the N-terminal HA tag, indicated as gray box) is cleaved by formic acid at the single Asp–Pro (DP) bond, generating two peptides of ~35 and 14 kDa. After extended incubation times, the two Asp–Gly (DG) bonds will also be cleaved, generating peptides of ~14, 21, 12, and 2 kDa. The proposed cleavage sites are indicated by arrowheads; the asterisk marks the two potential EPG modification sites at Glu289 and Glu362. B and C, T. brucei {Delta}procyclin#1 trypanosomes were incubated with 2.5 µCi/ml [3H]Etn for 23 h and extracted as described in the legend to Fig. 3. SDS extracts (1.6 x 106 cell equivalents) were incubated with a 7-fold volume of 70% formic acid for 0, 4, and 22 h, dried, and subsequently analyzed by SDS-PAGE and fluorography (B), or immunoblotting using {alpha}-EF antibody (C). The lanes contain 1.6 x 106 cell equivalents for fluorography and 1.8 x 105 cell equivalents for immunoblotting. D and E, T. brucei {Delta}procyclin#1 trypanosomes expressing HA-tagged eEF1A were labeled with 2.5 µCi/ml [3H]Etn for 23 h. After immunoprecipitation, HA-eEF1A was treated with 70% formic acid for 0, 4, and 22 h as above, and analyzed by SDS-PAGE and fluorography (D), or immunoblotting using {alpha}-HA antibody (E). The lanes contain 1.5 x 108 cell equivalents for fluorography and 2 x 107 cell equivalents for immunoblotting.

 
Although a decrease in [3H]Etn incorporation into PE is consistent with a decreased rate of PE synthesis by the Kennedy pathway, it may not reflect a decrease in cellular PE content, because, at least in mammalian cells and yeast, substantial amounts of PE are generated by decarboxylation of PS (22, 23). Thus, we determined the total PE content in T. brucei procyclic forms after down-regulation of ethanolamine kinase or ethanolamine-phosphate cytidylyltransferase by RNAi. The results show that knocking down the two enzymes has a dramatic effect on cellular PE levels. After 5 days of incubation in the presence of tetracycline, ethanolamine kinase knockdowns showed a 38% reduction in total PE, whereas ethanolamine-phosphate cytidylyltransferase knockdowns had <20% of PE compared with cells incubated in the absence of tetracycline (Fig. 8C). In contrast, the PC content was unaffected during down-regulation of PE biosynthesis (results not shown), indicating that the two enzymes that were targeted by RNAi are only involved in PE synthesis and show no significant overlapping substrate specificity with the corresponding enzymes of the Kennedy pathway for PC biosynthesis. Furthermore, these results demonstrate that PC in T. brucei is not synthesized by methylation of PE, which is in agreement with the apparent lack of genes for the corresponding methyltransferases in the T. brucei genome. Furthermore, the dramatic reduction of cellular PE after down-regulation of ethanolamine-phosphate cytidylyl-transferase demonstrates that the Kennedy pathway provides the majority of PE in T. brucei procyclic forms.


Figure 7
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 7.
Growth phenotype of RNAi cell lines targeting enzymes of the Kennedy pathway. Expression of putative ethanolamine kinase and putative ethanolamine-phosphate cytidylyltransferase in T. brucei procyclic forms was down-regulated by tetracycline-inducible double-stranded RNA. A, total RNA of cells after induction of RNAi by tetracycline (Tet) for 5 days was extracted and probed with oligonucleotides against the respective sequences by Northern blotting (top panels). rRNA was used as loading control (bottom panels). B, growth of ethanolamine kinase RNAi clones B1 (triangles) and D4 (circles) and ethanolamine-phosphate cytidylyltransferase RNAi clones A4 (squares) and B5 (diamonds) was monitored in the absence (filled symbols) or presence (open symbols) of tetracycline over a period of 10 days. The data points represent cumulative cell numbers.

 
Down-regulation of PE Synthesis Blocks EPG Attachment to eEF1A—Because PE has been shown to be the donor of the Etn residue for GPI anchor assembly in trypanosomes (46), incorporation of [3H]Etn into GPI-anchored EP procyclin was expected to be severely affected after down-regulation of PE synthesis and, thus, was used as positive control for a limited availability of PE for protein modification. The results show that, during labeling of procyclic T. brucei RNAi cells with [3H]Etn in the absence of tetracycline, significant amounts of radioactivity were incorporated into eEF1A and EP procyclin (Fig. 9A). In contrast, after RNAi-mediated down-regulation of PE synthesis, labeling of eEF1A and EP procyclin was partly (RNAi against ethanolamine kinase) or completely (RNAi against ethanolamine-phosphate cytidylyltransferase) inhibited (Fig. 9A). Immunoblotting of the same extracts shows that RNAi against the two enzymes did not reduce the amounts of eEF1A (Fig. 9B). In contrast, protein levels of EP were decreased in the induced cells compared with control uninduced trypanosomes (Fig. 9C). In addition, two bands with lower molecular masses were detected in cells after down-regulation of ethanolamine-phosphate cytidylyltransferase (Fig. 9C); these likely represent precursor forms of EP procyclin.


Figure 8
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 8.
Effects of ethanolamine kinase RNAi and ethanolamine-phosphate cytidylyltransferase RNAi on PE metabolism. T. brucei RNAi clone B1 against ethanolamine kinase and clone B5 against ethanolamine-phosphate cytidylyltransferase were cultured for 3 days in the absence (-) or presence (+) of tetracycline (Tet). A, to label PE, cells during the last 18 h of culture were incubated in the presence of 1.5 µCi/ml [3H]Etn. Subsequently, trypanosomes were washed to remove unincorporated label, and phospholipids were extracted with CM and separated by TLC using solvent system 1. The 3H-labeled lipids from 9 x 105 cell equivalents were visualized by scanning the individual lanes. The upper and lower panels show the results after RNAi against ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase, respectively. B, to label acid-soluble precursors of PE, cells during the last 2 h of culture were incubated in the presence of 0.5 µCi/ml [3H]Etn. Subsequently, trypanosomes were washed to remove unincorporated label, and the biosynthetic PE precursors were extracted with PCA and analyzed by TLC using solvent system 2. The 3H-labeled metabolites from 5 x 107 cell equivalents were visualized by scanning the individual lanes. The arrows indicate the site of sample application (O), the front of the solvent (F), and the migration of different standards run on the same TLC plate. PE, phosphatidylethanolamine; Etn-P, ethanolamine-phosphate; CDP-Etn, CDP-ethanolamine. C, to determine the total PE content of RNAi clones B1 and B5 cultured for 5 days in the absence (white bars) or presence (black bars) of tetracycline, phospholipids from 4 x 108 trypanosomes were extracted with CM and separated by TLC using solvent system 1. After visualization of lipids by iodine vapor, spots co-migrating with a PE standard run on the same plate were scraped and quantified by lipid phosphorous analysis. The values represent means ± S.E. from three independent experiments.

 


Figure 9
View larger version (56K):
[in this window]
[in a new window]

  		FIGURE 9.
Effects of PE down-regulation on EPG and GPI modification. T. brucei RNAi clone B1 against ethanolamine kinase and clone B5 against ethanolamine-phosphate cytidylyltransferase were cultured for 3 days in the absence (-) or presence (+) of tetracycline (Tet). During the last 18 h, trypanosomes were labeled with 1.5 µCi/ml [3H]Etn. Subsequently, the cells were washed and delipidated as described in Fig. 3. The resulting pellet was resuspended in 1% SDS and analyzed by SDS-PAGE and fluorography, or immunoblotting. A, labeling of eEF1A and EP procyclin by [3H]Etn. Each lane contains 1.2 x 108 cells equivalents. B and C, detection of eEF1A and EP procyclin using {alpha}-EF (B) and {alpha}-EP (C) antibody, respectively. The blot in B was stripped and re-probed in C. Each lane contains 2 x 106 cell equivalents.

 
To further demonstrate that the reduction of PE synthesis had a major effect on GPI anchor biosynthesis and attachment to protein, T. brucei procyclic forms after RNAi were labeled with [3H]Etn or [3H]myristate, and the GPI precursors and free GPI anchors were analyzed by TLC and SDS-PAGE followed by fluorography. The results show that the major GPI precursor lipid, named PP1 (42), was labeled with [3H]Etn in control cells but not in cells after induction of RNAi against ethanolamine-phosphate cytidylyltransferase (supplemental Fig. S2A). In contrast, when trypanosomes were labeled with [3H]myristate, the amount of 3H-labeled GPI precursor lipids increased considerably (supplemental Fig. S2B). Similarly, induced cells showed a depletion of [3H]Etn-labeled and an accumulation of [3H]myristate-labeled free GPI anchors (supplemental Fig. S2C). Together, these results are consistent with a depletion of Etn-containing GPI lipids and free GPIs and a concomitant accumulation of GPI lipids and free GPIs lacking the terminal Etn group in cells after RNAi against ethanolamine-phosphate cytidylyltransferase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that eEF1A in the parasitic protozoa, T. brucei, is modified with EPG. Labeling experiments, in combination with chemical and mass spectrometric analyses, show that, in analogy to mammalian cells (9–11) and plants (11), endogenous and HA-tagged eEF1A in T. brucei procyclic and bloodstream forms can be labeled with [3H]Etn at an EPG moiety attached to the side chain of a glutamate residue. This amino acid, Glu362 in the T. brucei eEF1A primary sequence, is strictly conserved between the predicted amino acid sequences of mammalian, plant, and yeast cells. In contrast, in the yeast S. cerevisiae, the corresponding Glu372 has been reported not to be modified with EPG (12). The reason of this lack of EPG addition to eEF1A in S. cerevisiae is unknown. Mammalian cells (9, 10) and plants (11) contain a second EPG modification attached to Glu301 or Glu289, respectively, of eEF1A (see also Fig. 2). In contrast, we found that the corresponding Glu289 of T. brucei eEF1A is not labeled with [3H]Etn. In addition, an EPG-modified tryptic peptide covering the second potential EPG site could not be detected by mass spectrometry. Therefore, it is likely that Glu289 of T. brucei eEF1A is not modified with EPG, which distinguishes it from all previously identified EPG-modified eEF1As. We were unable to identify an amino acid sequence motif that predicts EPG attachment to one or both glutamate residues in eEF1A of the different organisms. Point mutations at and around the potential modification sites are currently underway to define the requirements for EPG attachment to eEF1A.

The pathways for the assembly of the EPG modification and its attachment to eEF1A have not been studied in detail before. We now show using RNAi against the first two enzymes of the Kennedy pathway that PE is donor of the Etn moiety for EPG in T. brucei eEF1A. We found that down-regulation of ethanolamine kinase or ethanolamine-phosphate cytidylyltransferase causes a severe reduction in total cellular PE content, demonstrating that the Kennedy pathway is the major route for PE synthesis in T. brucei procyclic culture forms. Importantly, inhibition of this pathway results in a partial (RNAi against ethanolamine kinase), or complete (RNAi against ethanolamine-phosphate cytidylyltransferase) block in incorporation of [3H]Etn into eEF1A, demonstrating that PE is a direct precursor of the EPG modification. This conclusion is supported by our findings that Etn or Etn-P cannot serve as precursors, because a block in PE synthesis by RNAi against ethanolamine-phosphate cytidylyltransferase completely prevents [3H]incorporation into eEF1A. These results are in contradiction to a previous report showing that Etn is directly added to eEF1A in a cell-free system (47). In addition, experiments targeting the third step in PE synthesis by the Kennedy pathway showed that RNAi against a putative ethanolamine-phosphotransferase also led to a reduction in [3H]incorporation into eEF1A, indicating that CDP-Etn is not a direct precursor for EPG (results not shown).

In addition, we show that a block in PE synthesis by the Kennedy pathway also results in a reduction, or lack, of [3H]Etn incorporation into GPI-anchored EP procyclin. This result is consistent with a previous report showing that PE is the donor of the Etn moiety linking the GPI anchor to protein in procyclic form trypanosomes and yeast (46, 48), and, thus, inhibition of PE synthesis is expected to inhibit GPI anchor attachment to procyclins. To our knowledge, this is the first time that GPI anchoring of proteins has been blocked by inhibition of an enzyme that is not directly involved in the GPI biosynthetic pathway.

Recent advances in the structure determination of eEF1A have clarified its role during protein translation elongation (5). In contrast, although eEF1A contains a number of (post-translational) modifications, their roles in eEF1A function are largely unknown. Amino acid modifications of eEF1A include phosphorylation, lysine methylation, and methyl esterification at the C terminus. Similarly, nothing is known about the role of EPG in eEF1A function. The available three-dimensional structures of yeast eEF1A (4, 5) and the high homology between eEF1As from different organisms predict the EPG-modified glutamate residues in mammalian cells, plants, and T. brucei to be located at the surface of the third polypeptide domain of eEF1A. This allows us to propose the following mechanism for EPG attachment to eEF1A. During or shortly after protein synthesis, eEF1A is modified with PE at one or more of the conserved glutamate residues in the third domain. This modification occurs at, or near, the site of PE synthesis and temporarily renders eEF1A membrane-bound. Interestingly, plant eEF1A has been reported to incorporate 14C-labeled fatty acids, however, no direct evidence was provided that eEF1A was modified with an entire PE molecule (11). Similar experiments in T. brucei procyclic forms to label eEF1A with [3H]myristate or [3H]palmitate, or by incubating the cells with [3H]pyruvate, which is incorporated into the fatty acid pool in T. brucei procyclic forms (49), showed no incorporation of radioactivity into eEF1A (results not shown). However, this is not surprising, because we were unable to detect a PE-modified form of eEF1A by mass spectrometry, and, thus, this form may represent a minor fraction of total eEF1A only. After addition of PE to eEF1A, the acyl and/or alkyl (or alkenyl) chains of PE will be removed by the concerted action of esterases of the phospholipase A-type and/or glyceryl ether-cleaving enzymes, respectively, releasing eEF1A to the cytosol. Such a pathway offers interesting implications for eEF1A function, because a small fraction of eEF1A may associate with membranes and could be involved in processes other than its canonical role in protein synthesis at the ribosome, such as tRNA import into mitochondria (8), actin-binding (7), association with sphingosine kinases (50) or other signaling molecules (51), or binding to peroxisomes (52).


   FOOTNOTES
 
* The work was supported by the Swiss National Science Foundation (Grant 3100A0-116627). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1. Back

1 To whom correspondence should be addressed. Tel.: 41-31-631-4113; Fax: 41-31-631-3737; E-mail: peter.buetikofer{at}mci.unibe.ch.

2 The 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. Back


   ACKNOWLEDGMENTS
 
We thank I. Roditi and A. Schneider for plasmids and cell lines and M. A. J. Ferguson for help in performing and interpreting the mass spectrometry experiments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Negrutskii, B. S., and El'skaya, A. V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47-78[Medline] [Order article via Infotrieve]
  2. Merrick, 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, NY
  3. Browne, G. J., and Proud, C. G. (2002) Eur. J. Biochem. 269, 5360-5368[Medline] [Order article via Infotrieve]
  4. Andersen, G. R., Valente, L., Pedersen, L., Kinzy, T. G., and Nyborg, J. (2001) Nat. Struct. Biol. 8, 531-534[CrossRef][Medline] [Order article via Infotrieve]
  5. Andersen, G. R., Nissen, P., and Nyborg, J. (2003) Trends Biochem. Sci. 28, 434-441[CrossRef][Medline] [Order article via Infotrieve]
  6. Condeelis, J. (1995) Trends Biochem. Sci. 20, 169-170[CrossRef][Medline] [Order article via Infotrieve]
  7. Gross, S. R., and Kinzy, T. G. (2005) Nat. Struct. Mol. Biol. 12, 772-778[CrossRef][Medline] [Order article via Infotrieve]
  8. Bouzaidi-Tiali, N., Aeby, E., Charriere, F., Pusnik, M., and Schneider, A. (2007) EMBO J. 26, 4302-4312[CrossRef][Medline] [Order article via Infotrieve]
  9. Rosenberry, T. L., Krall, J. A., Dever, T. E., Haas, R., Louvard, D., and Merrick, W. C. (1989) J. Biol. Chem. 264, 7096-7099[Abstract/Free Full Text]
  10. Whiteheart, S. W., Shenbagamurthi, P., Chen, L., Cotter, R. J., and Hart, G. W. (1989) J. Biol. Chem. 264, 14334-14341[Abstract/Free Full Text]
  11. Ransom, W. D., Lao, P.-C., Gage, D. A., and Boss, W. F. (1998) Plant Physiol. 117, 949-960[Abstract/Free Full Text]
  12. Cavallius, J., Zoll, W., Chakraburtty, K., and Merrick, W. C. (1993) Biochim. Biophys. Acta 1163, 75-80[CrossRef][Medline] [Order article via Infotrieve]
  13. Ferguson, M. A. J. (1999) J. Cell Sci. 112, 2799-2809[Abstract]
  14. Eisenhaber, B., Maurer-Stroh, S., Novatchkova, M., Schneider, G., and Eisenhaber, F. (2003) BioEssays 25, 367-385[CrossRef][Medline] [Order article via Infotrieve]
  15. Kinoshita, T., and Inoue, N. (2000) Curr. Opin. Chem. Biol. 4, 632-638[CrossRef][Medline] [Order article via Infotrieve]
  16. Orlean, P., and Menon, A. K. (2007) J. Lipid Res. 48, 993-1011[Abstract/Free Full Text]
  17. Kabeya, Y., Mizushima, N., Yamamoto, A., Oshitani-Okamoto, S., Ohsumi, Y., and Yoshimori, T. (2004) J. Cell Sci. 117, 2805-2812[Abstract/Free Full Text]
  18. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and Ohsumi, Y. (2000) Nature 408, 488-492[CrossRef][Medline] [Order article via Infotrieve]
  19. Nakatogawa, H., Ichimura, Y., and Ohsumi, Y. (2007) Cell 130, 165-178[CrossRef][Medline] [Order article via Infotrieve]
  20. Dixon, H., and Williamson, J. (1970) Comp. Biochem. Physiol. 33, 111-128[Medline] [Order article via Infotrieve]
  21. Patnaik, P. K., Field, M. C., Menon, A. K., Cross, G. A. M., Yee, M. C., and Bütikofer, P. (1993) Mol. Biochem. Parasitol. 58, 97-106[CrossRef][Medline] [Order article via Infotrieve]
  22. Vance, J. E. (2003) Prog. Nucleic Acids Res. Mol. Biol. 75, 69-111[Medline] [Order article via Infotrieve]
  23. Birner, R., and Daum, G. (2003) Int. Rev. Cytol. 225, 273-323[Medline] [Order article via Infotrieve]
  24. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193-214[Free Full Text]
  25. Vance, J. E., and Vance, D. E. (2004) Biochem. Cell Biol. 82, 113-128[CrossRef][Medline] [Order article via Infotrieve]
  26. Vance, J. E., and Steenbergen, R. (2005) Prog. Lipid Res. 44, 207-234[CrossRef][Medline] [Order article via Infotrieve]
  27. Choi, J. Y., Wu, W. I., and Voelker, D. R. (2005) Anal. Biochem. 347, 165-175[CrossRef][Medline] [Order article via Infotrieve]
  28. Vial, H. J., Eldin, P., Tielens, A. G. M., and van Hellemond, J. J. (2003) Mol. Biochem. Parasitol. 126, 143-154[CrossRef][Medline] [Order article via Infotrieve]
  29. van Hellemond, J. J., and Tielens, A. G. (2006) FEBS Lett. 580, 5552-5558[CrossRef][Medline] [Order article via Infotrieve]
  30. Vassella, E., Bütikofer, P., Engstler, M., Jelk, J., and Roditi, I. (2003) Mol. Biol. Cell 14, 1308-1318[Abstract/Free Full Text]
  31. Wirtz, E., Leal, S., Ochatt, C., and Cross, G. A. (1999) Mol. Biochem. Parasitol. 99, 89-101[CrossRef][Medline] [Order article via Infotrieve]
  32. Bütikofer, P., Vassella, E., Ruepp, S., Boschung, M., Civenni, M., Seebeck, T., Hemphill, A., Mookherjee, N., Pearson, T. W., and Roditi, I. (1999) J. Cell Sci. 112, 1785-1795[Abstract]
  33. Ruepp, S., Furger, A., Kurath, U., Kunz Renggli, C., Hemphill, A., Brun, R., and Roditi, I. (1997) J. Cell Biol. 137, 1369-1379[Abstract/Free Full Text]
  34. Bochud-Allemann, N., and Schneider, A. (2002) J. Biol. Chem. 277, 32849-32854[Abstract/Free Full Text]
  35. Chomczynski, P., and Sacchi, N. (2006) Nat. Protoc. 1, 581-585[CrossRef][Medline] [Order article via Infotrieve]
  36. Bütikofer, P., Ruepp, S., Boschung, M., and Roditi, I. (1997) Biochem. J. 326, 415-423[Medline] [Order article via Infotrieve]
  37. Field, 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, Oxford
  38. Bütikofer, P., Boschung, M., Brodbeck, U., and Menon, A. K. (1996) J. Biol. Chem. 271, 15533-15541[Abstract/Free Full Text]
  39. Rifkin, M. R., Strobos, C. A. M., and Fairlamb, A. H. (1995) J. Biol. Chem. 270, 16160-16166[Abstract/Free Full Text]
  40. Rouser, G., Fleischer, S., and Yamamoto, A. (1970) Lipids 5, 494-496[Medline] [Order article via Infotrieve]
  41. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  42. Field, M. C., Menon, A. K., and Cross, G. A. M. (1991) EMBO J. 10, 2731-2739[Medline] [Order article via Infotrieve]
  43. Roditi, I., and Liniger, M. (2002) Trends Microbiol. 10, 128-134[CrossRef][Medline] [Order article via Infotrieve]
  44. Inglis, A. S. (1983) Methods Enzymol. 91, 324-332[Medline] [Order article via Infotrieve]
  45. Acosta Serrano, A., Cole, R. N., and Englund, P. T. (2000) J. Mol. Biol. 304, 633-644[CrossRef][Medline] [Order article via Infotrieve]
  46. Menon, A. K., Eppinger, M., Mayor, S., and Schwarz, R. T. (1993) EMBO J. 12, 1907-1914[Medline] [Order article via Infotrieve]
  47. Whiteheart, S. W., and Hart, G. W. (1994) Arch. Biochem. Biophys. 309, 387-391[CrossRef][Medline] [Order article via Infotrieve]
  48. Menon, A. K., and Stevens, V. L. (1992) J. Biol. Chem. 267, 15277-15280[Abstract/Free Full Text]
  49. Stephens, J. L., Lee, S. H., Paul, K. S., and Englund, P. T. (2007) J. Biol. Chem. 282, 4427-4436[Abstract/Free Full Text]
  50. Leclercq, T. M., Moretti, P. A., Vadas, M. A., and Pitson, S. M. (2008) J. Biol. Chem. 283, 9606-9614[Abstract/Free Full Text]
  51. Panasyuk, G., Nemazanyy, I., Filonenko, V., Negrutskii, B., and El'skaya, A. V. (2008) Int. J. Biochem. Cell Biol. 40, 63-71[CrossRef][Medline] [Order article via Infotrieve]
  52. Kiel, J. A., Titorenko, V. I., van der Klei, I. J., and Veenhuis, M. (2007) FEMS Yeast Res. 7, 1114-1125[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Signorell, M. Rauch, J. Jelk, M. A. J. Ferguson, and P. Butikofer
Phosphatidylethanolamine in Trypanosoma brucei Is Organized in Two Separate Pools and Is Synthesized Exclusively by the Kennedy Pathway
J. Biol. Chem., August 29, 2008; 283(35): 23636 - 23644.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/29/20320    most recent
M802430200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Signorell, A.
Right arrow Articles by Bütikofer, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Signorell, A.
Right arrow Articles by Bütikofer, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement