The Tandem PDZ Protein Syntenin Interacts with the Aminoacyl tRNA

The Tandem PDZ Protein Syntenin Interacts with the Aminoacyl tRNA Synthetase Complex in a Lysyl-tRNA Synthetase-Dependent Manner Kris Meerschaert,†,‡,# Eline Remue,†,‡ Ariane De Ganck,†,‡ An Staes,†,‡ Ciska Boucherie,†,‡ Kris Gevaert,†,‡ Joe¨l Vandekerckhove,†,‡ Lawrence Kleiman,§ and Jan Gettemans*,†,‡ Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium, Department of Biochemistry, Ghent University, Faculty of Medicine and Health Sciences, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium, and Lady Davis Institute for Medical Research and McGill AIDS Center, Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2 Received April 29, 2008

Syntenin-1 is a tandem PDZ protein that binds a diverse array of signaling molecules that are often associated with cell adhesion and intracellular trafficking. With the use of a MS-based functional proteomics approach, we identified several members of the aminoacyl-tRNA synthetase macromolecular (ARS) complex in a syntenin-1 pull down assay. Interaction of these proteins with syntenin-1 was confirmed by co-immunoprecipitation from cultured cells. We demonstrate a direct interaction of syntenin-1 with lysyl-tRNA synthetase (KRS), which contains a PDZ binding motif at its C-terminus. This motif is important for the interaction of the entire complex with syntenin-1. A point mutation in the PDZ2 domain of syntenin-1 abrogates interaction with KRS. As a result, other components of the ARS complex no longer co-immunoprecipitate with syntenin-1. We further show that syntenin-1 regulates KRS activity. These findings suggest that syntenin-1 is an adaptor modulating the activity of KRS. Keywords: PDZ • Syntenin • Signal transduction • Functional proteomics • tRNA synthetase

Introduction Proteins containing PDZ domains function as molecular scaffolds in the formation and organization of multiprotein complexes at the membrane, connecting cell adhesion molecules, ion channels and receptors to downstream signaling proteins. PDZ proteins play important roles in several cellular and biological processes such as the establishment and maintenance of cell polarity and cell adhesion, and the regulation of cell growth, development, and differentiation.1,2 PDZ domains are ubiquitous protein-interaction modules that in most cases recognize the C-terminal part of their binding partners which binds in a pocket formed by the PDZ domain’s second β-strand, second R-helix, and linker that precedes these secondary structure elements, called the carboxylate binding loop. PDZ domains have been typically grouped in different classes depending on the targeted C-terminal sequence, but this classification has been questioned.3,4 * To whom correspondence should be addressed. Dr. Jan Gettemans, Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium, and Department of Biochemistry, Ghent University, Faculty of Medicine and Health Sciences, Albert Baertsoenkaai 3, B-9000 Ghent, Belgium. Tel: 00 32 9 2649340. Fax: 00 32 9 2649490. E-mail: [email protected]. † Department of Medical Protein Research, VIB. ‡ Ghent University. # Current address: Ablynx nv, Technologiepark, 9052 Ghent/Zwijnaarde, Belgium. § Jewish General Hospital.

4962 Journal of Proteome Research 2008, 7, 4962–4973 Published on Web 10/08/2008

Syntenin-1 has two tandem PDZ domains and was originally identified as a gene down-regulated during human melanoma differentiation5 and as a protein that binds directly to the cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans.6 Syntenin-1 functions in a variety of biological processes such as transcription, vesicular trafficking, receptor clustering and syndecan recycling, cell adhesion and migration and the regulation of membrane-cytoskeleton interactions.7-11 Syntenin-1 also plays a role in pathological processes since it has been shown to be overexpressed in breast and gastric cancer cells and in melanoma, where it promotes the migration and metastasis of cancer cells.12-15 These diverse biological functions are a result of its high number of interacting partners which include syndecans,6 various glutamate receptors,16-18 the transcription factor sox4,7 the serine/threonine kinase Unc51.1 and rab5,19 ephrin ligands and ephrin receptor tyrosine kinases,20 pro-transforming growth factor-alpha,8 PTPη,21 neurofascin,22 neurexin,23 merlin,24 Frizzled 7,25 IL-5 receptor alpha,6 CD626 and CD63.27 In most cases, these proteins associate with syntenin-1 through direct interaction with its PDZ domains. Interestingly, the tandem PDZ domains of syntenin-1 and its close homologue syntenin-2 also constitute a phosphoinositide binding domain,28,29 and we have previously shown that phosphatidylinositol 4,5-bisphosphate (PIP2) binding, as well as receptor binding, regulates the targeting of syntenin-1 to the plasma membrane. Lipid binding also regulates the recycling of syndecan to the plasma mem10.1021/pr800325u CCC: $40.75

 2008 American Chemical Society

Tandem PDZ Protein Syntenin Interacts with the ARS 10

brane and the invasion-inducing properties of syntenin through collagen.15 The crystal structure of the tandem PDZ domains of syntenin-1 in complex with different target peptides has been elucidated.30 The two PDZ domains have a typical PDZ fold with two antiparallel β-sheets capped by two R-helices. A crystal structure of the single PDZ-2 domain31 showed that different target sequences could interact with this domain by the differential use of three distinct subsites, which could explain the degenerate binding specificity of syntenin-1. Although several interaction partners for syntenin-1 have been described in different cell types, we set out to identify new binding partners for syntenin-1 using a mass spectrometrybased approach. In this way, we identified tRNA synthetases in a GST-syntenin-1 pull down assay. These tRNA synthetases are part of the multisynthetase (ARS) complex and are involved in catalyzing the ligation of amino acids and tRNAs in the process of translation. Lysyl-tRNA synthetase (KRS) was subsequently identified as a direct syntenin-1 binding partner. The association between these proteins is mediated by the Cterminus of KRS and full length syntenin-1. Both proteins coimmunoprecipitate from cell extracts. Our results identify KRS as a new syntenin-1-binding protein and unveil a function for syntenin-1 as a regulator of KRS activity.

Materials and Methods Reagents. Polyclonal affinity purified anti-syntenin antibody was from Synaptic Systems (#133 003), rabbit polyclonal antiKRS antiserum was from Abcam, 9E10 anti-Myc antibody from Upstate Cell Signaling Solutions and rabbit anti-GFPs were obtained from Santa Cruz and Upstate Cell Signaling Solutions. For immunoprecipitations of GFP and GFP fusion proteins, an in-house affinity purified polyclonal antibody was used (kindly donated by Dr. V. De Corte). All commercial antibodies were used at the dilution recommended by the manufacturer. Custom made rabbit polyclonal antibodies for human lysRS, MetRS, GlnRS, TrpRS, ArgRS, TyrRS, ProRS, IleRS, anti-p43, and anti-p38 were used as previously described.32 The expression plasmids pGEX and pEGFP containing fulllength (FL), PDZ 1 + 2C, PDZ-1, PDZ-2 and the N-terminal domain of syntenin-1 and mutants therein were described previously.15 Myc tagged full-length (1-597 amino acids) human KRS cloned into pcDNA3.1 (Invitrogen, Merelbeke, Belgium) and 6×His-tagged KRS into pET21B were described previously.33 Proteins. Expression of recombinant His6-tagged KRS was induced with 0.1 mM IPTG in TOP10 cells and cells were grown overnight at 20 °C. KRS was purified on Probond Ni2+Sepharose beads (Invitrogen) according to the manufacturer’s instructions. Protein concentrations were estimated by the Bradford method using the Bio-Rad protein assay kit and bovine serum albumin as a standard.34

Procedures Cell Culture and Tissue Extracts. HEK293T cells were maintained at 37 °C in a humidified 10% CO2 incubator and grown in Dulbecco’s modified Eagle’s medium + Glutamax (DMEM; Invitrogen), supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin and 0.1 mg/mL streptomycin. MCF-7 AZ breast cancer cells were maintained in a mixture (1/1) of HAM F12 (Invitrogen) and DMEM + Glutamax also supplemented with 10% FBS, 100 U/mL penicil-

research articles lin and 0.1 mg/mL streptomycin. Placental extracts were obtained by homogenizing the tissue in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton-X100, and 1 mM PMSF buffer using a Dounce homogenizer followed by 5 min sonication. The extract was centrifuged at 40 000g for 1 h. Fresh protein material was used for pull down experiments. GST Pull Down and Immunoprecipitation. For GST pull down assays, 10 µg of the individual GST fusion proteins bound to glutathione-agarose beads (Amersham Biosciences) were incubated at 4 °C for 1 h with 1-2 mg of cell lysate (HEK293T, MCF-7). A total of 10-15 mg of soluble protein from placenta was used for each pull down experiment. Following incubation with the glutathion-Sepharose immobilized fusion proteins, the resin was washed three times with lysis buffer, boiled in Laemmli sample buffer and electrophoresed by SDS-PAGE. For immunoprecipitations, 1-2 µg of affinity purified rabbit anti-GFP antibody was incubated with 1-1.5 mg of cell lysate overnight at 4 °C followed by immobilization on 20 µL of settled protein G-Sepharose beads (Amersham Biosciences) for 1 h. The beads were washed three times with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton-X100, and 1 mM PMSF), and bound proteins were eluted in Laemmli sample buffer followed by separation on SDS-10% PAGE and immunoblotting. Western Blotting. Cells were disrupted in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton-X100, and 1 mM PMSF) and a protease inhibitor cocktail mix (Roche Diagnostics, Mannheim, Germany). Insoluble material was removed by centrifugation (20 000g for 15 min at 4 °C). Western blotting was performed as described.35 Proteins were visualized by enhanced chemiluminescence detection (ECL, Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Mass Spectrometry. Protein bands of interest were cut from the gel and transferred to Biopure Eppendorf tubes (Eppendorf AG, Hamburg, Germany). Following two 15 min washes with water/acetonitrile (1/1, v/v) (both Baker HPLC analyzed, Mallinckrodt Baker B.V., Deventer, The Netherlands), gel pieces were dried in a centrifugal vacuum concentrator and subsequently rehydrated in 10 µL of a 0.02 µg/µL sequencing grade modified trypsin stock solution (Promega Corporation, Madison, WI). Following rehydration, excess trypsin solution was removed and freshly prepared 50 mM NH4HCO3 (pH ) 7.9) was added to completely submerge the gel pieces. Trypsin digestion proceeded overnight at 37 °C and was stopped by acidification with formic acid. Peptide mass fingerprinting was performed on a Bruker Ultraflex II TOF/TOF MALDI mass spectrometer (Bruker Daltonics, Bremen, Germany). Briefly, 2 µL of the peptide mixture was spotted on a MTP AnchorChip 600/384 T F (Bruker Daltonics) and air-dried. Peptides were then incorporated into MALDI-matrix by adding 0.6 µL of a freshly prepared solution of 0.1 mg/mL R-cyano-4-hydroxycinnamic acid in ethanol/ acetone/0.1% TFA (6/4/1). After this solution was dried, samples were washed by briefly submerging the entire plate in 10 mM ammonium phosphate after which excess of this solution was removed under a gentle nitrogen stream. The MALDI mass spectrometer was operated as described previously.36 Peptide mixtures that were analyzed by LC-ESI-MS/MS were dried in a centrifugal vacuum concentrator and redissolved in 20 µL of 0.1% formic acid in water/acetonitrile (98/2, v/v) (HPLC solvent A). Half of each mixture was used for nanoLCMS/MS analysis on a Waters CapLC (Waters Corporation, Journal of Proteome Research • Vol. 7, No. 11, 2008 4963

research articles Milford, MA) in-line coupled to a Q-TOF mass spectrometer (Micromass UK Limited, Cheshire, U.K.). The sample was first trapped on a trapping column (PepMap C18, 0.3 mm i.d. × 5 mm from LC-Packings, Amsterdam, The Netherlands), and following elution by back-flushing, it was loaded on a 75 µm i.d. column (PepMap C18, LC-Packings). Following a 5 min wash step with solvent A, peptides were eluted with a linear gradient of 4% solvent B (0.1% formic acid in water/acetonitrile (3/7, v/v)) increase per min at a flow rate of 200 nL/min. With the use of data-dependent acquisition, doubly or triply charged ions with intensities above a threshold of 40 were selected for fragmentation. During MS/MS, a cone voltage of 30 V and a scan time of 1 s with an inter scan time of 0.07 s were used. The exclusion time for an ion mass was set to 200 s. Datadependent acquisition started 15 min following the start of the gradient. Peptide MS/MS fragmentation spectra were converted to pkl files using the Masslynx software (version 3.5, Waters Corporation) and were searched using MASCOT (http://www.matrixscience.com) against the Swiss-Prot database with restriction to human proteins. Peptide mass and fragment tolerances were set to 0.3 Da, and ESI-QUAD-TOF was the selected instrument for peptide fragmentation rules. Variable modifications were set to methionine oxidation, pyro-glutamate formation of amino terminal glutamine, deamidation of glutamine, and acetylation of the N-terminus. MS/MS-spectra that obtained a score exceeding Mascot’s identity threshold score at the 95% confidence level were kept and such identified peptides were stored in a MySQL relational database (http://genesis.ugent.be/ ms_lims/) and are here reported. Aminoacylation Assays. Enzymatic reactions were carried out as described.37 Briefly, assays were performed at 37 °C in 50 µL of reaction buffer containing 50 mM HEPES, pH 7.5, 20 mM KCl, 10 mM MgCl2, 20 mM β-ME, 3 mM ATP, 40 U/mL bovine liver tRNA (Sigma), 5 U/mL yeast inorganic pyrophosphatase (Sigma) and 20 µM L-lysine. Recombinant purified KRS was used at a final concentration of 140 nM. After 15 min, 100 µL of the Biomol Green reagent (Biomol Plymouth Meeting, PA) was added to the mixture to stop the reaction and to detect free inorganic phosphate produced by the pyrophosphatase from the pyrophosphate released during amino acid activation. After 10 min incubation at room temperature, the absorbance at 620 nm was measured using a Multiscan EX microplate reader (Thermo Labsystems). Reactions were performed in the absence or presence of GST or GST-FL syntenin-1, used at an equimolar ratio.

Results Identification of the Multiaminoacyl-tRNA Synthetase Complex by MS Analysis of Syntenin-1-Associated Proteins. Syntenin-1 contains 2 PDZ domains that are capable of interacting with different ligands. To search for new syntenin-1-binding proteins, we used a proteomic-based approach using a GSTfusion protein of wild-type syntenin-1 (GST-FL WT) as an affinity probe. Initially, GST-FL WT syntenin-1 was incubated with or without a Triton-soluble extract of human placenta. Bound proteins were eluted from the beads and visualized by SDS-PAGE (Figure 1A). Several bands were specifically retained by full-length syntenin-1 (compare lane 7 with lane 8) but not by the syntenin-1 PDZ1 domain (compare lanes 1 and 2), the PDZ2 domain (lanes 3-4) or by GST alone (lanes 5-6). Some of the proteins that were specifically retained by GST-FL WT syntenin-1 could be identified by MALDI-MS peptide-mass 4964

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Figure 1. Members of the ARS complex from placental extracts are retained by GST-FL syntenin-1. (A) Coomassie-stained gel of proteins isolated from placenta extracts, with the numbers corresponding to the identified proteins shown in Table 1. GSTtagged proteins were incubated without (lanes 1, 3, 5, 7) or with the extract (lanes 2, 4, 6, 8) and eluted from the beads using SDSsample buffer. The asterisk marks GST from placental extracts. The extract was preincubated with glutathione-Sepharose in the case of PDZ2. (B) Coomassie-stained gel of pulled down proteins from HEK293T (left) and MCF7 (right) extracts. GST (lane 1) and syntenin FL (lane 3) in the absence of extract or in the presence of cell extract (lanes 2 and 4, respectively). Proteins identified in the GST-FL syntenin-1 eluates are shown in Table 2. The positions of molecular mass standards (in kilodaltons) are shown to the left of each gel in (A) and (B). (C) Western blot with antiKRS antibody showing detection of KRS in MCF7 lysates (input) and after pull down with GST-FL syntenin-1.

fingerprinting and are given in Table 1. Interestingly, four out of the five identified proteins are tRNA synthetases that are part of a macromolecular multiaminoacyl-tRNA synthetase complex (ARS), which consists of nine different synthetases and three nonenzymatic auxiliary protein factors (ARS-interacting multifunctionalprotein):AIMP1/p43,AIMP2/p38,andAIMP3/p18.38,39 We repeated these experiments with Triton-soluble extracts from HEK 293T and MCF-7 cultured cells (Figure 1B). Following

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Tandem PDZ Protein Syntenin Interacts with the ARS a

Table 1. List of Syntenin-1-Associated Proteins Identified in a Placental Extract band

accession

protein

mascot score

sequence coverage

1 2 3 4 5

P07814 P41252 P55072 P47897 Q15046

Bifunctional aminoacyl-tRNA synthetase Isoleucyl-tRNA synthetase Transitional endoplasmic reticulum ATPase Glutaminyl-tRNA synthetase Lysyl-tRNA synthetase

115 87 98 102 95

11% 10% 17% 16% 17%

a Following in-gel trypsin digestion, pulled down proteins were identified by MALDI-MS peptide mass fingerprinting. Their Swiss-Prot accession number, Mascot protein score and the overall protein sequence coverage are indicated.

Table 2. List of Syntenin-1-Associated Proteins Identified in HEK293T and MCF7 Cell Extracts Following LC-ESI-MS/MS Analysisa

a

identified protein (+ isoform)

MCF7 peptides (coverage)

HEK peptides (coverage)

6-phosphofructokinase type C (Q01813) 6-phosphofructokinase, liver type (P17858) 78 kDa glucose-regulated protein precursor (P11021) Anti-colorectal carcinoma heavy chain (Q65ZQ1) Arginyl-tRNA synthetase, cytoplasmic (P54136) Aspartyl-tRNA synthetase, cytoplasmic (P14868) ATP-dependent RNA helicase A (Q08211) ATP-dependent RNA helicase DDX3X (O00571, O15523) Bifunctional aminoacyl-tRNA synthetase (P07814) Carbonyl reductase [NADPH] 1 (P16152) Drebrin (Q16643) Elongation factor 1-gamma (P26641) Glutaminyl-tRNA synthetase (P47897) Heat shock 70 kDa protein 1 (P 08107) Heat shock cognate 71 kDa protein (P11142) Heat shock protein HSP 90-beta (P08238) Heat shock-related 70 kDa protein 2 (P54652, P1142) Hetergeneous nuclear ribonucleoprotein A1 (P09651) Heterogeneous nuclear ribonucleoprotein A3 (P51991) Heterogeneous nucledar ribonucleoprotein H (P31943) Heterogeneous nuclear ribonucleoprotein U (Q00839) Heterogeneous nuclear ribonucleoproteins A2/B1 (P22626) Heterogeneous nuclear ribonucleoproteins C1/C2 (P07910) Heterogeneous nuclear ribonucleoproteins C1/C2 (P07910) Isoleucyl-tRNA synthetase, cytoplasmic (P41252) Leucyl-tRNA synthetase, cytoplasmic (Q9P2J5) Lysyl-tRNA synthetase (Q15046) Methionyl-tRNA synthetase, cytoplasmic (P56192) Multisynthetase complex auxiliary component p38 (Q13155) Multisynthetase complex auxiliary component p43 (Q12904) Myosin-VI (Q9UM54) Nuclease sensitive element- binding protein 1 (P67809) Protein phosphatase 1 regulatory subunit 12A (O14974) RNA-binding protein EWS (Q01844) RNA-binding protein FUS (P35637) Stress-70 protein, mitochondrial precursor (P38646) Syntenin-1 (O00560) Transitional endoplasmic reticulum ATPase (P55072)

12 (18%) 18 (28%) 2 (4%) N.D. 10 (18%) N.D. N.D. N.D. 23 (20%) 10 (50%) 4 (10%) 2 (5%) 11 (17%) N.D. 3 (5%) 2 (3%) N.D. 2 (7%) 3 (13%) 6 (18%) 2 (2%) 5 (15%) 4 (14%) 1 (16%) 18 (17%) 4 (4%) 16 (34%) 7 (9%) 2 (11%) 4 (15%) 12 (10%) 2 (8%) 4 (4%) 3 (6%) 6 (16%) 9 (18%) 2 (10%) 17 (24%)

N.D. N.D. N.D. 2 (5%) 17 (28%) 10 (22%) 7 (6%) 2 (4%) 20 (16%) N.D. N.D. N.D. 20 (31%) 2 (4%) N.D. N.D. 3 (6%) 2 (7%) 3 (13%) 6 (18%) 10 (16%) 5 (15%) 4 (14%) 1 (16%) 15 (12%) 5 (4%) 13 (26%) N.D. N.D. 4 (15%) N.D. N.D. N.D. 3 (6%) 6 (16%) 9 (18%) 2 (10%) N.D.

Their Swiss-Prot accession number, the number of identified peptides and the overall sequence coverage are indicated.

nano-LC-ESI-MS/MS analysis of in-gel tryptic digests from the excised protein bands, several tRNA synthetases were again identified as specifically associated with full-length syntenin-1 (Table 2). Western blotting with anti-lysyl tRNA synthetase (KRS) antibodies further confirmed the presence of this enzyme in the GST-syntenin-1 precipitate (Figure 1C). MS/MS spectra that were linked to lysyl tRNA synthetase are shown in Figure 2. Interaction between syntenin-1 and endogenous components of the ARS complex was further investigated by expression of EGFP-tagged syntenin-1 constructs in HEK293T cells (schematically shown in Figure 3A) followed by immunopre-

cipitation with anti-GFP antibodies. Western blotting using specific antibodies revealed that glutamyl-tRNA synthetase (QRS), isoleucyl-tRNA synthetase (IRS), lysyl-tRNA synthetase (KRS) and arginyl-tRNA synthetase (RRS) co-precipitated with full-length syntenin-1, but not with the N-terminal region of syntenin-1 nor with a syntenin-1 PDZ1 + 2C construct that lacked the N-terminal region (Figure 3B). Furthermore, the nonenzymatic components of the complex including p43 and p38 are also specifically co-immunoprecipitating with fulllength syntenin-1. The corresponding Coomassie stained gel is shown in the right part of Figure 3B. Examination of the C-terminal end of all the human aminoacyl tRNA synthetases Journal of Proteome Research • Vol. 7, No. 11, 2008 4965

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Figure 2. MS/MS spectra illustrating the identification of lysyl-tRNA-synthetase as a binding partner of syntenin-1. Three different peptide MS/MS spectra are given (panels A-C) and all b and y type of fragment ions present in these spectra are indicated.

in the complex revealed that there was only one member of the ARS complex, the lysyl tRNA synthetase that contains a PDZ binding motif at its C-terminus (VGTSV), which is a canonical class I PDZ binding motif. Therefore, we investigated if KRS could represent the direct link between the ARS complex and syntenin-1. Syntenin-1 Interacts with KRS through a PDZ Mediated Association. To examine the possibility that lysyl-tRNA synthetase interacts with syntenin-1 via its PDZ domains, we cotransfected EGFP-tagged wild-type syntenin-1 or a syntenin-1 mutant (‘K+S 8’) with myc-tagged KRS in HEK293T cells. The syntenin-1 mutant contains 8 point mutations in the tandem PDZ domains (K119A, 171SDK-HEQ173 in PDZ1 and K203A, 250KDS-SHE252 in PDZ2) that abrogate interaction with target ligands.23 For comparison, EGFP-PDZ1 + 2C and EGFP-Nterm were also expressed in HEK293T cells (Figure 4A). Syntenin-1 was immunoprecipitated with an anti-GFP antibody, and association with overexpressed KRS was determined by Western 4966


blotting. The Myc-KRS construct was equally expressed in all cases as determined by Western blotting on cell lysates (Figure 4A, top panel). However, a Myc-KRS signal was only apparent in immunoprecipitates with full-length syntenin-1 and MycKRS did not co-immunoprecipitate with mutant syntenin-1, the N-terminal region of syntenin-1 or syntenin-1 lacking the N-terminal region (Figure 4A, bottom panels). This observation indicates that only full-length syntenin-1 is able to bind KRS and that both its N-terminal domain and the tandem PDZ domains are involved. PDZ domains usually interact with target ligands through binding with their carboxy terminal sequence. We deleted the last 4 amino acids of KRS and expressed this construct (KRS dPBM) deleted in its PDZ-binding motif) in HEK293T cells together with syntenin-1 to investigate if the interaction was abolished. Immunoprecipitates were analyzed by Western blotting with GFP antibodies (syntenin-1) or Myc antibodies (KRS). Figure 4B shows that while wild-type KRS interacts with

Tandem PDZ Protein Syntenin Interacts with the ARS

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Figure 3. Co-immunoprecipitation of enzymatic and nonenzymatic components of the ARS complex with FL syntenin-1. (A) Schematic representation of GFP-tagged FL syntenin-1 and its deletion mutants. Indicated are the amino acids that correspond to the different constructs that were used (see Procedures). (B) HEK293T cells were transfected with pEGFP vector or pEGFP-tagged syntenin-1 constructs. Coprecipitating tRNA synthetases were detected in the anti-GFP immunoprecipitates and in the lysates (input ) 5% of total protein incubated) by Western blotting using specific antibodies against glutamyl-tRNA synthetase (QRS), isoleucyl-tRNA synthetase (IRS), lysyl-tRNA synthetase (KRS), arginyl-tRNA synthetase (RRS) and p43 and p38. A coomassie stained gel showing the immunoprecipitated syntenin-1 constructs is depicted on the right.

EGFP-syntenin-1 (lane 3), this interaction is strongly reduced in the KRS mutant (lane 4), suggesting that association between syntenin-1 and KRS occurs via the carboxy-terminal end of the tRNA synthetase. No association was observed between wildtype or mutant KRS and GFP (lanes 1-2). Syntenin-1 contains two PDZ domains, and to investigate which domain is responsible for the interaction, we used two previously described syntenin-1 mutants in which the last glycine residue in the carboxylate-binding loop of each PDZ domain was mutated to glutamate (G128E in PDZ1) or aspartate (G212D in PDZ2).22 This glycine is necessary for the interaction with the ligand’s carboxylate group.40 The mutation in the first domain (G128E) only caused a minor reduction of the binding to KRS as observed in a co-immunoprecipitation experiment (Figure 4C, lane 3). In contrast, the G212D construct, as well as the double mutant (G128E/G212D), failed to interact with KRS (Figure 4C, lanes 4 and 5). This indicates that the second PDZ domain is required to bind KRS. The syntenin-1 double mutant G128E/G212D allowed us to further investigate if other selected components of the aminoacyl tRNA synthetase complex also interact directly with syntenin-1, for example, through a noncanonical PDZ binding.

To this end, we expressed the syntenin-1 G128E/G212D mutant in HEK293T cells and analyzed co-immunoprecipitation of various components of the ARS complex with this mutant (Figure 5). Importantly, although IRS, KRS, RRS and p43 coimmunoprecipitated with wild-type syntenin-1 (Figure 5, lane 2), none of these components associated with GFP (Figure 5, lane 1) or with mutant syntenin-1 (Figure 5, lane 3). These results indicate that the other components of the aminoacyl tRNA synthetase complex indirectly associate with syntenin-1 through the lysyl tRNA synthetase subunit of the complex. Syntenin-1 Modulates KRS Enzymatic Activity. The aminoacylation reaction catalyzed by KRS, as well as other tRNA synthetases, is a two step reaction involving hydrolysis of ATP into PPi and coupling of AMP to the amino acid (amino acid activation). In the next step, the activated amino acid is transferred to its cognate tRNA. In view of the direct association between KRS and syntenin-1, we analyzed a possible regulation of KRS activity by syntenin-1 by measuring the first step of the aminoacylation reation (ATP hydrolysis, see Materials and Methods) in a colorimetric assay allowing quantification of the amount of inorganic phosphate released during the enzymatic reaction. Recombinant KRS was purified to homogeneity Journal of Proteome Research • Vol. 7, No. 11, 2008 4967

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Figure 4. KRS co-immunoprecipitates with wild-type FL syntenin-1, not with mutant syntenin-1. (A) HEK293T cells were co-transfected with pEGFP-tagged syntenin-1 constructs and Myc-tagged KRS. Co-precipitating KRS was detected in the anti-GFP immunoprecipitate and in the crude lysate (input ) 5% of total protein incubated) by Western blotting using monoclonal anti-Myc antibody. The same material was probed with a polyclonal anti-GFP antibody (lower diagrams) to verify the presence of GFP or GFP fusion proteins. (B) HEK293T cells co-transfected with GFP or GFP-FL syntenin-1 and Myc-tagged KRS WT or a mutant deleted in the last 4 amino acids (KRS dPBM). Co-precipitating KRS was detected in the anti-GFP immunoprecipitate and in the crude lysate (input ) 5%) by Western blotting using anti-Myc antibodies. The same blot was incubated with anti-GFP antibody (lower diagrams) to verify the presence of GFP or GFP fusion proteins. (C) HEK293T cells were co-transfected with pEGFP-tagged syntenin-1 WT and mutant constructs and Myc-tagged KRS. Co-precipitating KRS was detected in the anti-GFP immunoprecipitate and in the crude lysate (input) by Western blotting using anti-Myc antibodies.

(Figure 6A) and incubated with or without GST (control) or GST-syntenin-1. We found that GST alone had no significant effect on KRS activity (Figure 6B). In contrast, GST-syntenin1, when used at an equimolar ratio to KRS, considerably inhibited KRS activity (approximately 40%, Figure 6B). Endogenous Syntenin-1 Interacts with KRS. To determine if the interaction between syntenin-1 and KRS also occurs in vivo between the endogenous proteins, we performed coimmunoprecipitations on a HEK293T cell extract with antisyntenin-1, anti-KRS or control rabbit IgG antibodies. Following immunoprecipitation of syntenin-1 and immunoblotting for KRS, we observed KRS in the syntenin-1 immunoprecipitate (Figure 7A, lane 3). However, in the reverse experiment in which KRS was immunoprecipitated, we could not detect syntenin-1 (Figure 7A, lane 2), indicating that perhaps the antibodies used for immunoprecipitating KRS interfere with syntenin-1 binding. Neither syntenin-1 nor KRS was precipitated using the negative 4968


control anti-rabbit IgG (Figure 7A, lane 1). The input represents 5% of total protein used in the immunoprecipitation. Syntenin-1 Binds Directly to KRS. The experiments described above suggest a direct interaction between syntenin-1 and KRS. To rule out the possible participation of an intermediate protein, we analyzed the KRS/syntenin-1 interaction in a GST pull down assay with recombinant proteins. GST or GSTFL syntenin-1-loaded glutathione beads were incubated with an extract from E. coli cells expressing His6-tagged human KRS. Figure 7B shows that His6-KRS interacts with GST-syntenin-1 but not with GST, indicating that KRS interacts directly with syntenin-1.

Discussion In this study, a number of potentially new syntenin-1 binding partners were identified using a full length GST-tagged syntenin-1 construct to pull down proteins for MS identification.


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Figure 5. Other components of the ARS complex do not independently associate with syntenin-1 but co-precipitate by virtue of KRS. GFP, GFP-wild-type syntenin-1 or the GFP-syntenin G128E/G212D mutant were transfected in HEK293T and recovered with anti-GFP antibodies. Various ARS components co-precipitate with wild-type syntenin-1 (left, lane 2) but not with GFP (left, lane 1) or mutant syntenin-1 (left, lane 3). The same blot was incubated with anti-GFP antibody (lower diagrams) to verify the presence of GFP or GFP fusion proteins. IgGH) GFP immunoglobulin heavy chain. Equal expression of the constructs was verified by Western blotting on a cytosolic extract (right, input ) 16%).

Figure 6. Syntenin-1 regulates KRS activity. Recombinant KRS with or without GST-syntenin-1 were used to assay the first step of the aminoacylation reaction. Pyrophosphate released is hydrolyzed by the pyrophosphatase and free inorganic phosphate is measured colorimetrically at 620 nm. Data were obtained from 3 independent experiments. Enzymatic activity was estimated based on the amount of phosphate released after 15 min reaction time: KRS (1.89 ( 0.16 nmol), KRS + GST (1.78 ( 0.12 nmol), KRS + GST-syntenin-1 (1.15 ( 0.10 nmol). KRS was used at 140 nM and GST-syntenin-1 was included at the same concentration.

Among the candidates, we established lysyl tRNA synthetase (KRS) as a new cytoplasmic binding partner of syntenin-1 and showed that syntenin-1 is able to recruit other members of the multiaminoacyl-tRNA synthetase (ARS) complex through its binding to KRS. Syntenin-1 is a scaffolding protein involved in cell-cell and cell-matrix adhesion, and the regulation of plasma membrane dynamics. It probably also plays a role in nuclear processes9 and a putative link with translation has been suggested.41 Aminoacyl-tRNA synthetases (ARSs) are essential enzymes catalyzing the ligation of amino acids to their cognate tRNA in the process of translation thereby ensuring faithful translation of the genetic code. In higher eukaryotes, nine different enzymes form the ARS macromolecular complex together with three nonenzymatic factors. In addition to their enzymatic functions, many of the enzymes of the complex perform other

roles such as tRNA export, rRNA synthesis, apoptosis, inflammation and angiogenesis in mammalian cells. For instance, the glutaminyl-prolyl-tRNA synthetase interacts with the ribosomal subunit L13a and GAPDH which together form a gene silencing complex when the cells are exposed to IFN-gamma.42 Others, such as glutaminyl-tRNA synthetase and methionyl-tRNA synthetase are involved in the regulation of apoptosis through an interaction with ASK1 and rRNA biogenesis in the nucleolus, respectively.43,44 For KRS (lysyl-tRNA synthetase) there are also some noncanonical functions described. KRS has been shown to be involved in the regulation of MITF and USF2 transcription factors in mast cells by generating Ap4A45 and it is involved in the assembly and replication of HIV-1 through an interaction with the viral Gag protein.46 KRS is also secreted as a proinflammatory cytokine from human cells, and its secretion is induced by TNF-alpha.47 Secreted KRS binds to macrophages and peripheral blood mononuclear cells and enhances TNFalpha production in these cells and also their migration. The components of the multi-ARS complex are assembled and maintained in the complex in such a way that the different enzymes can carry out aminoacylation reactions simultaneously. AIMP2/p38 interacts with most of the ARS components, and is therefore proposed to be a scaffold for the assembly of the multi-ARS complex. However, other nonsynthetase factors, such as chaperone proteins, are also involved in facilitating the assembly of the complex. An interaction between heat shock protein 90 (hsp90) and the human glutamyl-prolyl-tRNA synthetase (EPRS), and other complexforming ARSs has recently been identified.48 Interestingly, we could find some HSP70 isoforms and HSP90 beta in our GSTpull downs, indicating that the presence of these proteins in the syntenin-1 pull down might be due to their interactions with the ARS complex. Furthermore, we also identified elongation factor 1-gamma in the syntenin-1 pull down in MCF7 cells, which has also been shown to be associated with the ARS complex.49 Next to aminoacyltransferases and associated components, we identified several other proteins that were specifically retained on FL syntenin-1 in contrast to a GST or single PDZ Journal of Proteome Research • Vol. 7, No. 11, 2008 4969

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Meerschaert et al. instance, we have identified a large number of heterogeneous nuclear ribonucleoproteins which were isolated from both HEK293T and MCF7 cells. These proteins bind to pre-mRNA, concomitant with transcription, and form ribonucleoprotein complexes essential for post-transcriptional events that range from mRNA packaging and transport, to mRNA splicing and silencing. Hence, hnRNP proteins play diverse roles both in the nucleus and cytoplasm, and regulate gene expression at various levels. Interestingly, two other RNA binding proteins EWS and FUS coprecipitate with syntenin-1. Syntenin-1 is partly localized in the nucleus9 and its homologue syntenin-2 has been shown to localize to nuclear speckles in a RNA dependent manner.29 We also identified the transitional endoplasmic reticulum ATPase (or valosin containing protein) both from placenta and from MCF7 cell extracts. VCP is implicated in the control of a variety of membrane functions, including membrane fusions, and is a regulator of the cell cycle. We further identified the enzyme carbonyl reductase in MCF7 cells as a possible syntenin-1 binding partner. However, this enzyme is known to bind covalently to GST via cysteine residues in its active site as reported before52 and is thus probably a nonspecific binder.

Figure 7. Endogenous syntenin-1 interacts with KRS and both proteins interact directly. (A) Western blot of immunoprecipitated proteins with anti-syntenin-1 and anti-KRS antibodies. One milligram of cell extract was incubated with the respective antibodies and probed for KRS or syntenin-1. Lane 1, immunoprecipitation with nonspecific rabbit IgG (1 µg); lane 2, immunoprecipitation with polyclonal antiserum to KRS (5 µL); lane 3, immunoprecipitation with polyclonal affinity purified antibody to syntenin-1 (1 µg); lane 4, lysate (input ) 5%) from HEK293T cells. (B) A ∼1 mg lysate of Escherichia coli cells expressing His6tagged KRS (lane 1, input ) 5%) was incubated with 10 µg of GST (lane 3) and GST-tagged FL syntenin-1 (lane 2) and then probed for KRS in the GST pull down using anti-KRS antibodies. A coomassie stainded gel showing the GST-tagged proteins is depicted below.

domain matrix (summarized in Table 2). Among these are also other proteins that contain putative PDZ-binding motifs (Table 2) and may bind, or have been shown to bind, PDZ proteins. For instance myosin VI, an actin-based molecular motor that translocates along actin filaments toward the minus end and which is implicated in endocytic trafficking, can bind to two different PDZ proteins, SAP97 and GIPC (synectin) through its C-terminal PDZ binding motif.50 Furthermore, an isoform of 6-fosfofructokinase (PFK-M) has been shown to interact with the PDZ domain of nNOS,51 while PFK-C also has a potential PDZ binding motif. However, further biochemical and molecular studies are required to determine their relationship with syntenin-1. Remarkably, we did not identify canonical binding partners of syntenin. However, in retrospect, this is not surprising considering the fact that most of the previously described binding partners are transmembrane proteins which are partially lost in our experiments in the Triton insoluble fraction. Also these proteins and several other identified partners are mostly of low abundance or expressed in a cell type specific manner. KRS is however abundantly and ubiquitously expressed. The relevance of the interaction of syntenin-1 with other proteins in table 2 not mentioned above is less clear. For 4970


Single PDZ domains could not bring down KRS in pull down experiments. This might suggest that the tandem PDZ domains are required to interact with KRS, as previously shown for several other ligands of syntenin. However, in this case the tandem PDZ domains are not sufficient and the interaction with KRS seems to depend on a full-length syntenin-1 protein since we could not observe an interaction with the PDZ1 + 2C construct, nor with a construct containing the N-terminal part. On the other hand, a single point mutation in the carboxylatebinding loop of PDZ2 disrupts the binding, indicating that an intact PDZ2 domain in the context of the full-length protein seems to be necessary. Significantly, other components of the ARS complex including IRS, RRS and p43 no longer associated with the syntenin-1 G128E/G212D double mutant, suggesting that they do not interact independently with syntenin-1 but co-immunoprecipitate due to their association with KRS. For PDZ1, a mutation in the carboxylate-binding loop reduces, but does not impair, the interaction between syntenin-1 and KRS, suggesting a less important role for this domain in the interaction. As such this is not unprecedented because most syntenin-1 ligands show preference for the PDZ2 domain.53 Interaction partners with a preference for the syntenin-1 PDZ1 domain include merlin, neurexin, CD63 (determined by using peptide ligands) and the recently identified Frizzled 7.25 As only full length syntenin-1 is capable of interacting with KRS, the syntenin-1 N-terminal domain must also play a role in the interaction. Although most ligand interactions involve both tandem PDZ domains of syntenin-1, interactions have even been reported that involve only the N-terminal region of syntenin-1, irrespective of its PDZ domains. Examples include eIF5A41 and the transcription factor SOX4.54 In the interaction between KRS and syntenin-1, the N-terminal domain of the latter may assist in the molecular recognition of KRS thus stabilizing the association. However, as a single point mutation in the PDZ2 domain suffices to abolish the interaction, the PDZ2 domain seems to be primarily responsible for KRS binding. Also, we cannot exclude that post-translational modifications in the N-terminal domain contribute to the interaction with KRS. Indeed, syntenin-1 harbors several potential tyrosine phosphorylation sites in its N-terminal domain53 and syntenin-1 has been reported to be tyrosine-phosphorylated when

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Tandem PDZ Protein Syntenin Interacts with the ARS 55

expressed in HEK293T cells. Alternatively, the N-terminal domain may affect KRS interaction through its tendency to promote self-association of the molecule. Koroll and coworkers22 showed that the interaction with PDZ2 of class I COOH termini with syntenin-1 lacking its N-terminal domain (PDZ1 + 2C) is abolished, whereas the binding of class II COOH termini to this mutant is retained. They demonstrated that this deletion construct did not self-associate and hypothesized that dimerization of syntenin-1 could be considered a prerequisite for interactions with class I but not with class II COOH-terminal peptides. The C-terminus of KRS (VGTSV) indeed constitutes a canonical class I PDZ binding motif. Moreover, several subunits of the ARS complex also form homo- and heterodimers including p43 and KRS.56,57 Oligomerization of syntenin-1, through its N-terminal domain, may be involved in modulating its interaction with KRS, in addition to PDZ2. The observation that syntenin-1 interacts with eIF5A41 as well as the multi-ARC complex through KRS (this paper) may point to a role as an adaptor protein regulating control of protein synthesis. In this respect, it is interesting to note that Methanothermobacter thermautotrophicus elongation factor-1R (EF1R) co-purifies with the archaeal multisynthetase complex (MSC) comprising LeuRS, LysRS and ProRS. Interactions between EF-1R and MSC contribute to translational fidelity by coupling aminoacylation of cognate tRNAs and their subsequent channeling to the ribosome.58 Hypothetically, syntenin-1 could act as a bridging protein between KRS (or ARS) and eIF5A and/or as an indirect modulator of the translation process. We showed that syntenin-1 partially inhibits hydrolysis of ATP into pyrophosphate and coupling of AMP to lysine (amino acid activation), which is the first step in the aminoacylation reaction catalyzed by KRS, indicating that sytenin-1 modulates KRS activity. Apart from small molecule inhibitors like the nonprotein amino acid S-(2-aminoethyl)-L-cysteine,59 thialysine and cadaverine,60 or compounds mimicking the lysyl adenylate complex,61 few KRS modulatory proteins have been described. Histidine triad nucleotide binding proteins (Hints), a superfamily of nucleotidyltransferases and hydrolyases, constitute a well documented example. Hint-AMP formation is dependent on formation of lysyl-AMP and lysyl-AMP acts as a substrate for Hints.62 Thus, Hints in a way regulate the activity of KRS by indirectly controlling the transfer of lysyl-AMP by KRS to its tRNA. While the mechanism of syntenin-1 inhibition of KRS remains to be elucidated, it is conceivable that syntenin-1 restrains the conformational flexibility of the enzyme by tight complex formation. Through this activity, syntenin-1 may also be involved indirectly in controling protein translation. The present findings also provide new avenues for exploring other physiological functions of syntenin-1, such as its possible role in the assembly and replication of HIV-1 through interaction between KRS and gag,63 and secretion of gag into exosomes, of which syntenin-1 is also a component,64 and as such, might also be involved in virus budding from infected cells. In addition, we and others have shown that syntenin-1 plays a role in cancer development by modulating invasion and metastasis of cancer cells.12-15 KRS is also highly enriched in tumor regions of breast cancer patients48 which may be related to the signaling properties of secreted KRS. Furthermore, both KRS secretion and syntenin-1 expression are stimulated by TNF-R.48,65 Thus, our findings raise the possibility of syntenin-1 being involved (as an adaptor molecule) in the secretion of KRS, owing to its role in the early secretory pathway8 or its possible

64

role in exosome release, and in this way promotes cancer cell invasion and metastasis. Abbreviations: KRS, lysyl tRNA-synthetase; ARS, aminoacyltRNA synthetase; AIMP, ARS-interacting multifunctional protein; FL, full length.

Acknowledgment. This work was supported by the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Concerted Actions Programme of Ghent University (GOA), the Interuniversity attraction poles (UAP06), and by the EU grant “Interaction Proteome” within the 6th Framework Program. K.M. was supported by a Postdoctoral Fellowship of the Fund for Scientific Research-Flanders (Belgium) (FWO-Vlaanderen). E.R. is supported by a fellowship from the research council of Ghent University (BOF). A.D.G. is supported by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). References (1) Harris, B. Z.; Lim, W. A. Mechanism and role of PDZ domains in signaling complex assembly. J. Cell Sci. 2001, 114 (Pt 18), 3219– 3231. (2) Jelen, F.; Oleksy, A.; Smietana, K.; Otlewski, J. PDZ domains common players in the cell signaling. Acta Biochim. Pol. 2003, 50 (4), 985–1017. (3) Bezprozvanny, I.; Maximov, A. Classification of PDZ domains. FEBS Lett. 2001, 509 (3), 457–462. (4) Vaccaro, P.; Dente, L. PDZ domains: troubles in classification. FEBS Lett. 2002, 512 (1-3), 345–349. (5) Lin, J. J.; Jiang, H.; Fisher, P. B. Melanoma differentiation associated gene-9, mda-9, is a human gamma interferon responsive gene. Gene 1998, 207 (2), 105–110. (6) Grootjans, J. J.; Zimmermann, P.; Reekmans, G.; Smets, A.; Degeest, G.; Durr, J.; David, G. Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. Proc. Natl. Acad. Sci. U.S.A. 1997, 94 (25), 13683–13688. (7) Geijsen, N.; Uings, I. J.; Pals, C.; Armstrong, J.; McKinnon, M.; Raaijmakers, J. A.; Lammers, J. W.; Koenderman, L.; Coffer, P. J. Cytokine-specific transcriptional regulation through an IL-5Ralpha interacting protein. Science 2001, 293 (5532), 1136–1138. (8) Fernandez-Larrea, J.; Merlos-Suarez, A.; Urena, J. M.; Baselga, J.; Arribas, J. A role for a PDZ protein in the early secretory pathway for the targeting of proTGF-alpha to the cell surface. Mol. Cell 1999, 3 (4), 423–433. (9) Zimmermann, P.; Tomatis, D.; Rosas, M.; Grootjans, J.; Leenaerts, I.; Degeest, G.; Reekmans, G.; Coomans, C.; David, G. Characterization of syntenin, a syndecan-binding PDZ protein, as a component of cell adhesion sites and microfilaments. Mol. Biol. Cell 2001, 12 (2), 339–350. (10) Zimmermann, P.; Zhang, Z.; Degeest, G.; Mortier, E.; Leenaerts, I.; Coomans, C.; Schulz, J.; N’Kuli, F.; Courtoy, P. J.; David, G. Syndecan recycling is controlled by syntenin-PIP2 interaction and Arf6. Dev. Cell 2005, 9 (3), 377–388. (11) Sarkar, D.; Boukerche, H.; Su, Z. Z.; Fisher, P. B. mda-9/syntenin: recent insights into a novel cell signaling and metastasis-associated gene. Pharmacol. Ther. 2004, 104 (2), 101–115. (12) Koo, T. H.; Lee, J. J.; Kim, E. M.; Kim, K. W.; Kim, H. D.; Lee, J. H. Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines. Oncogene 2002, 21 (26), 4080–4088. (13) Helmke, B. M.; Polychronidis, M.; Benner, A.; Thome, M.; Arribas, J.; Deichmann, M. Melanoma metastasis is associated with enhanced expression of the syntenin gene. Oncol. Rep. 2004, 12 (2), 221–228. (14) Boukerche, H.; Su, Z. Z.; Emdad, L.; Baril, P.; Balme, B.; Thomas, L.; Randolph, A.; Valerie, K.; Sarkar, D.; Fisher, P. B. mda-9/ Syntenin: a positive regulator of melanoma metastasis. Cancer Res. 2005, 65 (23), 10901–10911. (15) Meerschaert, K.; Bruyneel, E.; De Wever, O.; Vanloo, B.; Boucherie, C.; Bracke, M.; Vandekerckhove, J.; Gettemans, J. The tandem PDZ domains of syntenin promote cell invasion. Exp. Cell Res. 2007, 313 (9), 1790–1804. (16) Enz, R.; Croci, C. Different binding motifs in metabotropic glutamate receptor type 7b for filamin A, protein phosphatase 1C, protein interacting with protein kinase C (PICK) 1 and syntenin

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PR800325U

Journal of Proteome Research • Vol. 7, No. 11, 2008 4973

The Tandem PDZ Protein Syntenin Interacts with the Aminoacyl tRNA

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