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Eukaryotic Cell, November 2007, p. 1979-1991, Vol. 6, No. 11
1535-9778/07/$08.00+0     doi:10.1128/EC.00249-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Membrane-Bound eIF2 Kinase in the Flagellar Pocket of Trypanosoma brucei

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brazil,¹ Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland,² Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin³

Received 11 July 2007/ Accepted 3 September 2007

	ABSTRACT

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Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2 kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2 at Ser51. It also phosphorylates the highly unusual form of eIF2 found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2 kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2 kinase described in unicellular eukaryotes.

	INTRODUCTION

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Translational regulation plays a pivotal role in stress adaptation programs in eukaryotes, from yeast to mammals (10). Little is known about translational regulation in trypanosomatids, an early diverging group of organisms in the eukaryotic lineage. Trypanosomes encompass important human pathogens, such as Trypanosoma brucei, the agent of sleeping sickness in Africa, Trypanosoma cruzi, agent of Chagas’ disease in the Americas, and Leishmania spp., the agent of the devastating disease leishmaniasis (http://www.who.int/tdr). In their digenetic lifestyle, cycling between insects and mammals, trypanosomes are subjected to the stress of encountering the different physiological milieu of their new hosts and must adapt to these environments. Most gene regulation in these organisms relies on posttranscriptional mechanisms (8). This view is largely based on (i) the fact that their mRNAs are transcribed as polycistronic RNAs that are then trans-spliced with the addition of a 5' leader sequence, (ii) the lack of well-defined RNA polymerase II promoters, and (iii) the paucity of transcriptional regulators. Several examples have been described of mRNAs translated only in a particular stage of differentiation, in a mechanism involving regulatory 3'-untranslated region sequences (16, 34, 48). General translation may be regulated during the differentiation process in T. brucei, as indicated by drastic alterations in polysome profiles (5).

One of the most conserved stress-activated regulatory pathways in eukaryotes involves the inhibition of protein synthesis through the phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2) by a family of protein kinases that are activated by specific signals (10). eIF2, bound to GTP, delivers the initiator methionyl tRNA to the 40S ribosomal subunit in each round of translation initiation. For the formation of the 80S complex at the AUG initiator codon, GTP is hydrolyzed to GDP, with the release of eIF2-GDP. For a new cycle of initiation, GDP must be exchanged to GTP, in a process requiring the guanine exchange factor eIF2B. The phosphorylated form of eIF2 (eIF2-P) is a poor substrate for eIF2B but has a much higher affinity for eIF2B than the unphosphorylated eIF2-GDP, acting as a competitive inhibitor.

Whereas high levels of eIF2-P block translation, moderate levels of eIF2-P, while still allowing protein synthesis to take place, lead to translational activation of specific messages, such as GCN4 in Saccharomyces cerevisiae (25) and ATF4 in mammals (50). These messages contain upstream open reading frames (uORFs) in their leader sequences that inhibit the scanning ribosome from reaching the main ORF. Upon a reduction in the amounts of eIF2-GTP, as a result of the phosphorylation of eIF2, the ribosomes bypass the uORFs, and translate the main ORF. GCN4 and ATF4 are both bZIP transcriptional activators that regulate downstream responses aimed at alleviating the initial stress condition. GCN4 activates hundreds of genes involved in amino acid biosynthesis and intermediary metabolism (37). ATF4, besides regulating amino acid metabolism, also participates in other stress remedial programs (22). Thus, eIF2 phosphorylation can provide a means for both general repression of protein synthesis, as well as gene-specific translational activation.

GCN2, the sole eIF2 kinase in S. cerevisiae, is activated by amino acid deprivation and low levels of glucose or purine (24, 51). Mammals have three other eIF2 kinases in addition to GCN2: HRI, activated by the lack of heme; PKR, activated by double-stranded RNA and cytotoxic stresses; and PEK/PERK, activated by endoplasmic reticulum (ER) stresses (10, 20, 27-29, 53). Orthologs of PEK/PERK are found in Caenorhabditis elegans and Drosophila melanogaster and of HRI are found in Schizosaccharomyces pombe (44, 52). Recently, another eIF2 kinase was identified in Toxoplasma gondii that is activated under alkaline and temperature stresses (45).

Activation of these kinases is mediated by dimerization and autophosphorylation of specific residues in the catalytic region. This phosphorylation event allows the binding of the substrate (13). Activation is controlled by regulatory domains specific to each type of eIF2 kinase. For GCN2, the binding of uncharged tRNAs to a region with similarity to histidinyl tRNA synthetase (HisRS domain) promotes its activation (36, 40). In PEK/PERK, an ER transmembrane protein, the regulatory domain located in the lumen of the ER is normally bound to BiP; when unfolded proteins accumulate in the ER, BiP is released from the regulatory domain, which then dimerizes the protein, activating the cytoplasmic kinase domain (4, 32).

Given the relevance of eIF2 signaling in stress remedial programs in all eukaryotes, and the obvious relevance of posttranscriptional regulation in trypanosomatids, we approached this regulatory pathway in T. brucei. Here we describe the characterization of an eIF2 kinase of T. brucei that is a membrane-associated protein localized at the flagellar pocket. The data presented here suggest that this kinase has an important role in sensing the state of protein transport in these parasites.

	MATERIALS AND METHODS

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Yeast strains and growth conditions. Standard yeast methods were used (42). Yeast were grown in synthetic complete medium lacking amino acids to select for plasmids and supplemented with 2% glucose or with 2% raffinose or 10% galactose. The yeast strains used in the present study were as follows: H1643 (MATa ura3-52 leu2-3,-112 trp1-63 sui2 p1108[GCN4-lacZ] at trp1-63 [pSUI2 URA3]) (12), H1894 (MATa ura3-52 leu2-3,-112 trp1-63 gcn2), J80 (MATa ura3-52 leu2-3,-112 trp1-63 gcn2 sui2 [pSUI2 LEU2]), and J82 (MATa ura3-52 leu2-3,-112 trp1-63 gcn2 sui2 [pSUI2^S51A LEU2]) (11).

Parasite strains and growth conditions. T. brucei 427 (MITat 1.2) was used in all experiments. Procyclic forms were grown in SDM-79 medium supplemented with 10% fetal bovine serum at 28°C and bloodstream forms in HMI-9 medium supplemented with 10% fetal bovine serum and 10% Serum Plus (JRH Biosciences) at 37°C.

Cloning and plasmid constructions. Genomic DNA was isolated from T. brucei procyclic forms. PCR was performed with the indicated primers by using Taq DNA polymerase or Platinum Taq (Invitrogen). PCR products were cloned in TOPO, completely sequenced, and then transferred to the appropriate vectors. The entire coding sequence of TbeIF2 was obtained as two fragments, using the oligonucleotides BC340 (GGGGAGCTCATGGCAGCTTACGGT) and BC215R (GGGGAATTCTTCCCATGCGTGTTTACCG) for the sequence corresponding to amino acid residues 1 to 263 and oligonucleotides BC215F (GGGGGATCCTTTTATGAGGAAAAGTTACCA) and BC335 for residues 125 to 419. For expression in Escherichia coli, the complete sequence was obtained by using a ClaI site common to both fragments and introducing it into the SacI/NotI sites of pET28a(+), generating plasmid pBE495. For expression in yeast from the GAL1 promoter in a LEU2 plasmid, a EcoRI-BamHI fragment of pBM272 containing the GAL1 promoter was ligated with a BamHI-XbaI fragment of pBE495 containing the TbeIF2 sequence into the EcoRI/XbaI sites of pRS315 (43), generating plasmid pBE498. The expression of TbeIF2^125-419 in E. coli was obtained by cloning the PCR fragment from the TOPO plasmid into the BamHI/NotI sites of pET28a(+) (plasmid pBE501). For its expression from the GAL1 promoter in yeast, the NcoI(blunt)-NotI fragment from pBE501 was transferred into plasmid pBE498 digested with BamHI(blunt)-NotI, replacing the insert of the wild-type protein with the truncated one (plasmid pBE506). The construction of the mutation T169A was performed by PCR using the oligonucleotides BC215F (GGGGGATCCTTTTATGAGGAAAAGTTACCA) and BC400 (GGCACGAATGCGAATCCTAGCGATTTCCGT). The product digested with BamHI-TifI was used in a three-fragment ligation with fragment TifI-NotI from the wild-type TbeIF2^125–419 sequence and pET28a(+) linearized with BamHI-NotI (pBE520). For the expression of the mutant forms of Tb-eIF2^125-419 in yeast, the NcoI(blunt)-NotI fragment of pBE520 was cloned into pBE498 digested with BamHI(blunt)-NotI. The expression of yeast eIF2 under the control of GAL1 was obtained by transferring a BamHI-EcoRI blunted fragment from pBE280 (46) into the blunted BamHI site of pBE498. For the expression of the complete TbeIF2^T169A in E. coli, the AlwNI-NotI fragment of pBE520 and the SacI-AlwNI fragment of pBE495 were cloned into the pET28a(+) plasmid linearized with SacI-NotI, generating plasmid pBE587.

The kinase domain of TbeIF2K2 (residues 618 to 1009) was amplified with oligonucleotides BC442 (GGATCCATGCCACAACTCTTTC) and BC445 (GAATTCTCAGTTGGCTGACGG). The amplified product was transferred to pEGST (35) for expression in yeast as a fusion to glutathione S-transferase (GST) (pBE553). For raising antibodies, the putative regulatory domains of the kinases were cloned using the oligonucleotides BC436 (AAGCTTTTTCAAGTTGGAAGTCA) and BC437 (CTCGAGTTACTTCTTTCTCCG) for TbeIF2K1^1068-1236, BC438 (GAATTCATGCAACCATCAGGGG) and BC441 (AAGCTTTTATATTGGAATAGACTC) for TbeIF2K2^35-449, and BC446 (GGATCCATGGACTCTCAAAGTG) and BC450 (CTCGAGTTAATTAGAGATGTTCTC) for TbeIF2K3^1-476. The amplified products were transferred to pET28a(+).

The kinase domain of human PKR (amino acids 258 to 551) was transferred as a BamHI-HindIII(blunt) fragment from pC661-1 (30) into the pGEX2T plasmid linearized with BamHI-EcoRI(blunt), generating plasmid pBE527. The mouse eIF2 coding sequence was obtained from cDNA of brain total RNA by PCR with oligonucleotides BC398 (GGGGATCCATGCCGGGGCTAAGTTG) and BC399 (GGGAATTCTTAATCTTCGCTTTGGCTTCC). The amplified product was transferred to the plasmid pET28a(+), generating plasmid pBE526.

Total RNA from T. brucei was obtained by TRIzol extraction as described by the manufacturer (Invitrogen) and further purified by precipitation with 2 M LiCl. RNA was treated with DNase (1 U/µg), and 0.3 µg was used in the reaction with SuperScript One-Step RT-PCR using Platinum Taq (Invitrogen). The oligonucleotides used for the amplification of the sequences corresponding to the TbeIF2 mRNA were BC338 (AACGCTATTATTAGAACAGTTTCT) (spliced leader sequence) and BC334 (GGGTTGATGACCTTACCGATG).

Expression and purification of recombinant proteins. The soluble His₆-TbeIF2 wild-type and His₆-TbeIF2^T169A proteins expressed in E. coli BL21(DE3) were purified from cultures induced with 100 µM IPTG (isopropyl-ß-D-thiogalactopyranoside) at 23°C overnight on nickel chelating resin as described by the manufacturer (QIAGEN). The putative regulatory domains of the kinases TbeIF2K1, TbeIF2K2, and TbeIF2K3 fused with a N-terminal His tag were expressed in E. coli BL21(DE3). The insoluble His₆-TbeIF2K1^1068-1236 protein was purified from cultures induced with 1 mM IPTG at 37°C for 2 h by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The insoluble His₆-TbeIF2K2^35-449 protein was purified from cultures induced with 1 mM IPTG at 23°C overnight on nickel chelating resin as described by the resin manufacturer. The soluble His₆-TbeIF2K3^1-476 protein was purified from cultures induced with 1 mM IPTG at 30°C overnight on nickel chelating resin. The soluble His₆-eIF2 was purified from cultures induced with 400 µM IPTG at 23°C overnight. The soluble GST-PKR^KD (kinase domain) was purified from cultures induced with 400 µM IPTG at 23°C overnight on glutathione-Sepharose 4B as described by the resin's manufacturer (Amersham).

Preparation of cell extracts. Yeast cell extracts were prepared by agitation with glass beads in phosphate-buffered saline (PBS) containing 2 mM benzamidine, 1 µg of leupeptin/ml, 4 µg of aprotinin/ml, 10 µg of pepstatin A/ml, 1 µg of antipaine/ml, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, and 1 mM NaF. For T. brucei, parasites (ca. 5 x 10⁸ cells) were washed in PBS and resuspended in 150 µl of 20 mM Tris-HCl (pH 8.0)-150 mM NaCl-2 mM MgCl₂-2 mM EGTA-2 mM benzamidine-1 µg of leupeptin/ml-4 µg of aprotinin/ml-10 µg of pepstatin A/ml-1 µg of antipain/ml-1 mM phenylmethylsulfonyl fluoride-10 mM sodium pyrophosphate-1 mM NaF. After freezing and thawing, the suspension was centrifuged at 10,000 x g for 15 min. The supernatant was kept as soluble material. The pellet was resuspended in 150 µl of the same buffer supplemented with 1% Triton X-100. After centrifugation at 10,000 x g for 15 min, the supernatant was kept as the membrane-enriched fraction. Final insoluble material was solubilized in buffer containing 8 M urea.

Antisera and immunoblots. Antibodies were obtained in rabbits by immunization with the recombinant proteins purified from E. coli. Monospecific antibodies to His-TbeIF2K2^35-449 were obtained by adsorption to the purified recombinant His-TbeIF2K2^35-449 protein immobilized on a nitrocellulose filter, as described previously (39). For immunoblots, membranes were blocked with 5% nonfat milk in double-distilled H₂O for 1 h at 23°C, followed by incubation with primary antibodies for 1 h at 23°C or overnight at 4°C. The following conditions were used: (i) anti-TbeIF2 diluted 1:500 in PBS, (ii) purified anti-TbeIF2K2^35-449 diluted 1:500 in PBS, (iii) anti-(mammalian)eIF2 (Cell Signaling) diluted 1:1,000 in Tris-buffered saline (TBS)-0.1% Tween 20-5% bovine serum albumin (BSA), (iv) anti-(yeast)eIF2 (23) diluted 1:500 in PBS, (v) anti-eIF2-P (Biosource) diluted 1:1,000 in TBS-0.1% Tween 20-5% BSA, (vi) anti-GST (laboratory reagent, 1:5,000 in PBS), and (vii) anti-TbBiP (2) diluted 1:60,000 in PBS-0.1% Tween 20-5% BSA. After washes with 0.1% Tween 20 in PBS or TBS, bound antibodies were detected by using horseradish peroxidase (HRP)-protein A (Amersham Biosciences) diluted 1:4,000 in PBS for anti-TbeIF2, anti-TbeIF2K2^35-449, anti-GST, anti-(yeast)eIF2, anti-eIF2-P, and anti-BiP and by using HRP-conjugated goat anti-mouse immunoglobulin G (IgG; Santa Cruz Biotechnology, Inc.) diluted 1:4,000 in TBS-0.1% Tween 20-5% BSA for anti-(m)eIF2. The bound antibodies were detected by using enhanced chemiluminescence (Amersham Biosciences). When necessary, the antibodies were stripped off the membranes by incubation with 100 mM ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) at 50°C for 30 min.

Immunoprecipitation. Extract (700 µg) of induced J82 yeast strain carrying the plasmid pBE553 (GST-TbeIF2K2^KD) or pEGST (GST) was precleared by incubation with 20 µl of protein A-Sepharose CL-4B beads (Amersham Biosciences) and preimmune serum in buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 30 mM MgCl₂, and 1 mM NaF. The supernatant was incubated for 2 h at 4°C with 20 µl of protein A-Sepharose beads preincubated with anti-GST antibodies. The beads were washed three times with the same buffer, and the material bound to the beads used in the in vitro kinase assays.

In vitro phosphorylation assays. Kinase assays using purified PKR or GCN2 were performed as described previously (13). Phosphorylation reactions using the immunoprecipitated GST-TbeIF2K2^KD contained 30 µCi of [-³²P]ATP in buffer containing 4 mM Tris-HCl (pH 8.0), 50 mM imidazole, 120 mM NaCl, 1 mM EGTA, 1% Triton X-100, 2.5 mM MnCl₂, and 2 mM magnesium acetate in a volume of 30 µl. Reactions were allowed to proceed for 1 h at 23°C. Proteins were denatured in the absence of ß-mercaptoethanol (to avoid the comigration of the IgG heavy chain and TbeIF2), resolved by SDS-PAGE, and stained with Coomassie blue; the incorporation of radioactive phosphate into TbeIF2 was detected on a Typhoon phosphorimager.

Tomato lectin uptake and localization studies. Cultured bloodstream cells were washed with HEPES buffered-saline (50 mM HEPES, 50 mM NaCl, 5 mM KCl, 70 mM glucose [pH 7.5]) and resuspended at 10⁷/ml in HMI9-BSA (serum-free HMI9 medium supplemented with 0.5 mg of bovine serum albumin/ml) containing tomato lectin-biotin conjugate (TL; Vector Laboratories, Burlingame, CA) at 20 µg/ml. Cells were incubated at 5°C or 15°C for 30 min, followed by extensive washing with PBS. Washed cells were lightly prefixed with 0.1% formaldehyde and then fixed onto slides with methanol-acetone as described previously (1). Cells were then stained with Alexa 633-streptavidin (Molecular Probes, Eugene, OR) to detect bound and internalized TL. At the same time cells were also stained with rabbit anti-TbeIF2K2 and/or monoclonal anti-p67 (1) to detect the lysosome. Appropriate Alexa 488- and Alexa 633-conjugated goat anti-IgG (Molecular Probes) were used as secondary antibodies. Cells were also stained with 500 ng of DAPI (4',6'-diamidino-2-phenylindole)/ml. Serial image stacks (0.2-µm Z-increment) were collected at 100x (PlanApo oil immersion 1.4 NA) on a motorized Zeiss Axioplan IIi equipped with a rear-mounted excitation filter wheel, a triple-pass (DAPI-fluorescein isothiocyanate-Texas Red) emission cube, differential interference contrast optics, and a Orca AG charge-coupled device camera (Hamamatsu, Bridgewater, NJ). All images were collected with OpenLabs 5.0 software (Improvision, Inc., Lexington, MA), and fluorescence images were deconvolved by using a constrained iterative algorithm, pseudocolored, and merged by using Velocity 4.0 software (Improvision).

Dephosphorylation and deglycosylation assays. A total of 5 µg of total protein from the membrane-enriched fraction was incubated with 20 U of calf intestinal alkaline phosphatase (Promega) in buffer containing 50 mM Tris-HCl (pH 9.3 at 25°C), 1 mM MgCl₂, 100 µM ZnCl₂, and 1 mM spermidine in a volume of 10 µl at 37°C for 3 h. Deglycosylation was performed with the membrane-enriched protein fraction (20 µg) of procyclic parasites, denatured at 100°C for 5 min in buffer containing 50 mM sodium phosphate (pH 5.5), 0.1% SDS, and 50 mM ß-mercaptoethanol. After incubation on ice for 10 min, endoglycosidase H (Sigma) was added to the mixture, and the reaction was allowed to proceed for 18 h at 37°C.

	RESULTS

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Trypanosomatids encode three potential eIF2 kinases. Sequences for three potential eIF2 kinases are found in the genome of T. brucei, with similar counterparts in other two trypanosomatids, Trypanosoma cruzi and Leishmania major (3, 15, 26). We named these proteins TbeIF2K1 through TbeIF2K3. The alignment of the catalytic kinase domains (KD) of the T. brucei proteins and the known eIF2 kinases is shown in Fig. 1. The three proteins contain an insert between domains IV and V, typical of eIF2 kinases (21). The inserts range from 46 residues in TbeIF2K2 to 119 in TbeIF2K3. These sizes are within the range found for the eIF2 kinases, of which the extreme is the T. gondii TgKA, composed of 615 residues. TbeIF2K3 has another large insert located between domains VIB and VII, which is unusual for this class of kinases. An interesting feature of the T. brucei proteins is the site of putative autophosphorylation. As indicated by the boxed arrow in Fig. 1, TbeIF2K2 has a serine residue where all other kinases have a threonine residue. Phosphorylation of this residue in PKR promotes the recognition of the substrate (13). The presence of the serine residue in TbeIF2K2 was confirmed by DNA sequencing. Residue T487 of PKR, indicated with a boxed arrowhead, is an important determinant of specificity for recognition of the substrate. This residue is within an -helical domain that in eIF2 kinases is longer than in other class of kinases. This extended configuration, along with its orientation, determines the substrate specificity. This threonine is found in TbeIF2K2 and TbeIF2K3 but not in TbeIF2K1. The remaining residues that are typical of this class of kinases are for the most part conserved in the T. brucei proteins, as indicated by the arrowheads in the alignment of Fig. 1. Figure 2 depicts the putative regulatory domains of the three T. brucei kinases relative to their catalytic regions. TbeIF2K1 is apparently a GCN2 ortholog, containing a ring finger, WD repeat domain (RWD) and a HisRS-like domain, which in yeast GCN2 are involved in GCN1 and uncharged tRNA binding, respectively. The HisRS-like region of TbeIF2K1 was detected by the Phyre prediction engine (www.sbg.bio.ic.ac.uk/phyre). The domain architecture is similar to GCN2, with the RWD and HisRS-like regions flanking the kinase domain. No pseudokinase region was detected in TbeIF2K1. TbeIF2K2 and TbeIF2K3 putative regulatory domains have no similar counterparts in any database, except the other trypanosomatids orthologs. TbeIF2K2 has a predicted N-terminal signal sequence and a potential transmembrane domain, suggesting association with membranes and with a topological similarity to PEK/PERK, i.e., a type I transmembrane protein with a C-terminal cytosolic kinase domain and a large N-terminal ectoplasmic domain.

View larger version (81K): [in this window] [in a new window]   FIG. 1. T. brucei encodes three potential eIF2 kinases. The alignment of the catalytic domains of eIF2 kinases is shown. Identical residues are indicated in dark gray, and conserved residues are indicated in light gray. Subdomains I through XI are indicated above the sequences, as are the functional regions comprising the catalytic and activation loops. The kinase inserts between subdomains IV and V are indicated by dashes, with the number of residues in parentheses. The dots indicate conserved residues among kinases in general. Arrowheads indicate residues specific to the eIF2 kinases, with the boxed arrowhead indicating the substrate specificity determinant in PKR. The boxed arrow in subdomain VIII marks the residue in PKR that is autophosphorylated in the active protein. Sc, S. cerevisiae; Dm, D. melanogaster; Nc, N. crassa; Hs, H. sapiens; Sp, S. pombe; Rn, R. norvegicus; Tg, T. gondii; Ce, C. elegans. GeneDB accession numbers: TbK1, Tb11.02.5050; TbK2, Tb04.1H19.980; TbK3, Tb06.5F5.660. GenBank accession numbers: ScGCN2, NP_010569; DmGCN2, AAC47516; NcGCN2, CAA62973; HsGCN2, Q9P2K8; CePEK, Q19192; DmPEK, AAF61200; HsPEK, AAI26355; SpHRI, Q9UTE5; HsPKR, P19525; TgKA, AY518936.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Domain organization and expression of TbeIF2 kinases. (A) Schematics of the domain organization of the TbeIF2 kinases. Amino acid residue numbers are indicated above the bars, with the residues of the kinase domains (KD) corresponding to the sequences presented in Fig. 1. In TbeIF2K1, RWD indicates the ring finger, WD domain, and HRS indicates the sequence with similarity to histidinyl-tRNA synthetase. (B) Expression of the mRNA for each kinase. RT-PCR products obtained from procyclic (P) and bloodstream (B) forms, using the oligonucleotide pairs BC436-BC437 for TbeIF2K1, BC438-BC441 for TbeIF2K2, and BC446-BC450 for TbeIF2K3, with the expected sizes of 0.5, 1.2, and 1.4 kb, respectively; the controls represent the same DNase-treated RNA preparations used for RT-PCR, subjected to PCR with the primers for TbeIF2K1. (C) Expression of TbeIF2K2. Immunoblot of whole cells of bloodstream (B) and procyclic (P) parasites with anti-His6-TbeIF2K235-449 serum or with preimmune serum; the lane labeled "c" contains the purified His6-TbeIF2K235-449 protein that was used for immunization. The arrow indicates the TbeIF2K2 protein. Molecular weight standards (103) are indicated on the left panel.

TbeIF2K2 is localized to flagellar pocket and endosomal membranes. Indication of the membrane-bound nature of TbeIF2K2 was obtained by partial fractionation of cell extracts. Parasites were lysed in buffer lacking detergents, and the insoluble material was extracted in the same buffer containing 1% Triton X-100. As shown in Fig. 3A, virtually all TbeIF2K2 partitioned to the detergent-solubilized material. As a control, we probed the lower part of the same nitrocellulose membrane with antibodies directed against TbeIF2, a cytoplasmic protein (see below). Endoglycosidase H treatment of the membrane enriched fraction resulted in a decrease in size of TbeIF2K2, confirming the presence of N-glycans and thus its import and transit through the ER (Fig. 3B).

View larger version (8K): [in this window] [in a new window]   FIG. 3. TbeIF2K2 is a glycosylated membrane associated protein. (A) Partial fractionation. Procyclic forms were lysed by freeze-thawing and centrifuged as described in Materials and Methods. The soluble supernatant was separated (lane 1); the pellet was solubilized in the same buffer containing 1% Triton X-100, in the same volume as the supernatant. After centrifugation, the supernatant was separated (lane 2), and the pellet was solubilized in the same volume of buffer containing 8 M urea (lane 3). Identical volumes of the three samples were subjected to immunoblot analysis with anti-TbeIF2K235-449 antibodies (TbK2) and with anti-TbeIF2 serum (Tb-eIF2). (B) Endoglycosidase H treatment. Detergent-solubilized membrane fractions of procyclic parasites were treated with endoglycosidase H (+) or incubated under the same conditions without endoglycosidase H (–) and then subjected to immunoblotting with anti-TbeIF2K235-449 antibodies (TbK2).

View larger version (17K): [in this window] [in a new window]   FIG. 4. Localization of TbeIF2K2 in procyclic cells. (A) Affinity-purified anti-TbeIF2K235-449 antibodies. Immunoblot of membrane and soluble fractions obtained from procyclic parasites using monospecific purified antibodies used in the immunofluorescence assays. (B) Immunofluorescence of procyclic parasites. Immunofluorescence using affinity-purified anti-TbeIF2K235-449 antibodies (TbK2), followed by anti-rabbit IgG-fluorescein isothiocyanate. Nuclei were labeled with DAPI. The specificity of the signal was ascertained by preincubation of the antibody solution with the purified His6-TbeIF2K235-449 protein, prior to addition to the cells (bottom row).

View larger version (36K): [in this window] [in a new window]   FIG. 5. Localization of TbeIF2K2 in bloodstream cells. Bloodstream trypanosomes were incubated for 30 min with biotinyl TL at 5°C to allow flagellar pocket binding (A to D and E to H) or at 15°C to allow binding and uptake into the endosomal compartment (I to L, M to P, and Q to T) as described in Materials and Methods. Fixed and permeabilized cells were then stained with fluorescent streptavidin to detect bound or internalized TL (red), and as indicated with purified anti-TbeIF2K2 antibodies (TbK2) and or anti-p67 (middle panels). (A to H) Cells stained with anti-TbeIF2K2 (green) and anti-p67 (red). The discrete positioning of the anterior lysosome and the posterior flagellar pocket allow simultaneous imaging of these organelles in the same channel. (I to P) Cells stained with anti-TbeIF2K2 (green). (Q to T) Cells stained with anti-p67 (green). Merged DAPI/differential interference contrast images are presented in the leftmost panels with kinetoplast (k) and nucleus (n) labeled, and merged three-channel fluorescent images are presented in rightmost panels. The positions of the lysosome (Lys), endosome (Endo), and flagellar pocket (FP) are indicated. The arrowhead in panel H indicates a region of discrete TbeIF2K2 signal, presumably the endosome, that does not colocalize with the nearby flagellar pocket. Bars, 5 µm.

The kinase domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2.

View larger version (62K): [in this window] [in a new window]   FIG. 6. TbeIF2K2 phosphorylates specifically residue Ser51 in yeast and mammalian eIF2. (A) GST-TbeIF2K2KD (GST-TbK2) was expressed from the inducible GAL1 promoter in yeast strain J80, which contains the wild-type copy of the gene encoding eIF2, and in strain J82, which contains the mutant eIF2Ser51Ala. The growth of two independent transformants expressing GST-TbK2 is shown on raffinose- (left) and on galactose (right)-containing medium. As controls, the growth of the same strains expressing GST from the empty vector is shown (GST). (B) The expression of the proteins was determined by immunoblotting total cell extracts (5 µg of total protein) prepared from the same isolates as described above of strain J82 and of a strain without plasmid (), grown on galactose, using antibodies to GST. As a control, the blot was probed with antibodies against total yeast eIF2 (eIF2-T) after removal of the first antibodies (bottom panel). (C) The phosphorylation of the endogenous eIF2 was analyzed in strain J80 carrying the vector (GST) or the GST-TbeIF2K2KD expressing plasmid (GST-TbK2) after 4 or 8 h of induction with galactose, by immunoblotting with antibodies directed to the phosphorylated form of eIF2 (eIF2-P) and normalized with antibodies to total eIF2 (eIF2-T). (D) In vitro phosphorylation assays were performed with mammalian eIF2 purified from E. coli and with GST-TbeIF2K2KD immunoprecipitated from an extract of strain J82 grown in galactose, using as a control GST immunoprecipitated from the same strain carrying the empty vector. As a control for the efficiency of phosphorylation, we used GST-PKRKD purified from E. coli. Phosphorylation was detected by immunoblots with antibodies specific for eIF2-P and normalized with antibodies against total mammalian eIF2 (eIF2-T). The amounts of the GST fusion proteins in the reactions were determined by immunoblots of the upper portion of the gel using anti-GST antibodies (GST).

TbeIF2K2 phosphorylates TbeIF2 at T169.

View larger version (22K): [in this window] [in a new window]   FIG. 7. eIF2 in trypanosomatids. (A) Alignment of the N-terminal half of T. brucei and L. major eIF2 and other characterized eIF2 proteins. A line above the sequences indicates the unstructured loop encompassing the phosphorylated residue, indicated with an arrowhead. The structural domains of eIF2 are indicated with lines below the sequences. (B) Immunoblot of total cell extract of intact bloodstream (B) and procyclic (P) form parasites using anti-TbeIF2 serum; as a control, the lane labeled rTb2 contains the His6-TbeIF2 protein purified from E. coli.

View larger version (28K): [in this window] [in a new window]   FIG. 8. TbeIF2 substitutes for yeast eIF2. (A) Growth of yeast cells expressing TbeIF2. Derivatives of strain H1643 expressing the indicated eIF2 proteins from the GAL1 promoter were grown on plates containing synthetic medium supplemented with galactose in the presence or absence of 5-FOA. (B) Expression of TbeIF2125-419 in yeast. Total cell extracts (50 µg) of two isolates carrying TbeIF2125-419 and one isolate expressing yeast eIF2 selected from the 5-FOA plates and of control H1643 cells carrying only the vector were subjected to immunoblotting with anti-TbeIF2 serum; after stripping the first antibodies, the same membrane was incubated with antibodies against yeast eIF2. The bottom panel shows the filter stained with Ponceau S.

View larger version (24K): [in this window] [in a new window]   FIG. 9. In vitro phosphorylation of TbeIF2. Purified His6-TbeIF2 was used in in vitro reactions with purified GCN2 (left panels) and purified PKR (upper right panels) and with immunoprecipitated GST-TbeIF2K2KD (bottom right panels). Controls were purified yeast GST-eIF2 and GST-eIF2Ser51Ala for the GCN2 and PKR assays and purified His6-TbeIF2Thr169Ala for the TbeIF2K2 assay. Exposure times are indicated as "short" or "long." The total protein was visualized on the gels by Coomassie R250 stain.

View larger version (20K): [in this window] [in a new window]   FIG. 10. Modifications of TbeIF2K2 in bloodstream parasites. (A) Phosphorylation of TbeIF2K2. Membrane-enriched (mb) and soluble (sol) fractions (5 µg of total protein) of bloodstream (B) and procyclic (P) parasites were subjected to immunoblots with anti-TbeIF2K2 antibodies and with anti-BiP serum (left panels) (BiP partitions to both soluble and membrane enriched fractions). Membrane-enriched fractions from bloodstream (B) and procyclic (P) parasites were treated with calf intestinal alkaline phosphatase (CIAP), and subjected to immunoblot with anti-TbeIF2K2 antibodies (right panel). (B) TbeIF2K2 in high-density cultures of bloodstream forms. Membrane (mb) and soluble (sol) fractions (10 µg of total protein) from bloodstream forms grown to late logarithmic phase (2.4 x 106 cells/ml), and 12 h (2.0 x 106 cells/ml) or 24 h (4.0 x 105 cells/ml) later were subjected to immunoblotting with anti-TbeIF2K2 antibodies (TbK2) or anti-BiP (BiP) serum. The indicated number of cells corresponds to live parasites as determined by their motility at each time point.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We described here two highly unusual components of translational regulation found in T. brucei that seem to be conserved in T. cruzi and L. major. Considering that these organisms represent a deeply rooted branch of the eukaryotic kingdom, it is interesting that trypanosomatids have evolved an eIF2 subunit that is divergent from all other eukaryotes, regarding both the threonine residue in place of Ser51 and the unique N-terminal extension. Perhaps most surprising was the discovery of an eIF2 kinase that is membrane associated and completely constrained to a subcellular structure that is typical of this group of protozoa. Importantly, the data presented here clearly indicated that TbeIF2K2 is a kinase that phosphorylates eIF2 in a highly specific manner. Its substrate range, however, differs from the known eIF2 kinases. Although TbeIF2K2 was capable of phosphorylating yeast, mammalian, and the T. brucei eIF2, neither PKR nor GCN2 efficiently phosphorylated TbeIF2 in vitro. This difference is not due to the presence in TbeIF2 of a threonine in the position corresponding to serine 51 since both PKR and GCN2 can phosphorylate yeast or mammalian eIF2 at a threonine residue in place of S51 (31). The specificity of substrate recognition by PKR is given by a contiguous surface on one face of eIF2 comprising the S51 region; the kinase determinants G30, A31, and Met44; and the invariant peripheral docking site comprised by the KGYID motif, located 28 residues C terminal to the S51 residue (9). Of these, only residues G30 and M44 differ in the trypanosomatids’ protein, being replaced by serine and isoleucine, respectively. Mutations in both G30 and M44 abolish phosphorylation of eIF2 by PKR (14). These substitutions alone could then account for the poor ability of TbeIF2 to function as a substrate for PKR. However, other divergent residues in TbeIF2 in positions that are invariant in other eIF2 in this critical region may also hinder its recognition by PKR. TbeIF2K2 phosphorylation of mammalian, yeast, and T. brucei eIF2 suggests that the substrate-binding sites on this kinase can accommodate a wider range of residues.

Phosphorylation of eIF2 appears to be relevant as a mechanism of regulation of protein synthesis in these parasites as suggested by the existence of three eIF2 kinases, one of which we have clearly shown to be a bona fide eIF2 kinase, and by the presence of orthologs of the five subunits of the GTP exchange factor, eIF2B, of which the regulatory subunits , ß, and mediate the inhibitory effect caused by increased levels of eIF2-P in the cells. It is not clear at the moment whether the phosphorylation of TbeIF2 would result in a downstream regulatory signaling cascade, since there is an apparent lack of transcriptional factors of the bZIP type in trypanosomatids that could function as GCN4 or ATF4 (26). It seems then that this signaling may only involve the downregulation of general translation during specific situations. It is possible that proteins with no transcriptional role might be translated from messages containing uORFs when TbeIF2 is phosphorylated.

The predicted domain structure of TbeIF2K2 suggests that it is an integral membrane protein with an N-terminal luminal or ectoplasmic domain and a C-terminal cytoplasmic kinase domain. Our findings that it requires detergent for solubilization and that it is N glycosylated are consistent with this interpretation. Surprisingly, however, given its topological similarity to the metazoan ER kinase PEK/PERK, TbeIF2K2 is prominently localized to the flagellar pocket and closely associated endosomal membranes. The flagellar pocket localization was also confirmed by immunofluorescence assays with different antibodies raised against another region of the protein, encompassing the kinase domain (data not shown). The flagellar pocket is the only site where endocytosis and exocytosis occur in these parasites, and all proteins destined to the plasma membrane are directed to the flagellar pocket before reaching the parasite surface (19). Trypanosomes have a dense extracellular surface coat composed of variant surface glycoprotein that restricts macromolecular access to the plasma membrane; thus, the flagellar pocket functions as the primary portal for cross talk with the host. In this regard the predicted topology of TbeIF2K2, with a cytoplasmic kinase domain and a potential extracellular regulatory domain, is very suggestive of a role in sensory signaling. A limited repertoire of transmembrane proteins have been characterized in the general endomembrane system of bloodstream trypanosomes, including the lysosomal marker p67 (1), the endosomal marker membrane-bound acid phosphatase (17), and the invariant surface glycoproteins ISG65/75, which recycle between the endosome and cell surface (7). However, none of these is likely to have a role in sensory signaling. Bloodstream trypanosomes do have a flagellar membrane-associated adenylate cyclase with an architecture analogous to that of TbeIF2K2 and that is primarily localized to the flagellum membrane, but ligands for this potential receptor have never been identified (38).

Just what process may regulate TbeIF2K2 activity is currently uncertain, but its localization in the flagellar pocket suggests as one possibility the endocytic cargo load, which is greatly enhanced in the bloodstream stage of the life cycle (18). TbeIF2K2 is constitutively expressed but has a higher basal phosphorylation state in bloodstream parasites, and perhaps this regulates kinase activity which could, in turn, control endocytosis indirectly through translational attenuation of proteins destined to the cell membrane. Another possibility is that TbeIF2K2 activity is regulated during life cycle differentiation. In natural infections pleomorphic bloodstream parasites differentiate in a density-dependent manner from dividing long slender forms to nondividing short stumpy forms that are preadapted for transmission to the tsetse fly (33). Concomitant with this growth arrest phenotype, levels of protein synthesis drop dramatically (5). This "quorum-sensing" process is stimulated by a parasite-derived small molecule called stumpy induction factor (SIF) (49). Neither SIF nor its receptor have been identified, but the location of TbeIF2K2 and its ability to phosphorylate endogenous eIF2 and thereby regulate translation make it an attractive candidate receptor. The loss of TbeIF2K2 in high-density cultures of monomorphic bloodstream trypanosomes, which are resistant to SIF, may be related as a cause or a consequence to this process.

Another issue raised by our study is how an eIF2 kinase present exclusively in a particularly small area of the cell could regulate general translation. Protein synthesis in T. brucei is not restricted to any region of the cell, since ribosomes are spread in the cytoplasm, as seen in many published electron microscopy analysis. TbeIF2 is also found distributed in the cytoplasm, as judged by immunofluorescence analysis using the antibodies described here (data not shown). We can foresee two possibilities: (i) TbeIF2K2 may regulate localized protein synthesis in the vicinity of the flagellar pocket, or (ii) activated TbeIF2K2 may change cellular localization, for example, by being internalized via endocytic vesicles, with the catalytic domain thus gaining access to a large pool of cytoplasmic eIF2. This latter mechanism could perhaps account for the fraction of TbeIF2K2 found in endosomes and that found phosphorylated in bloodstream cells.

The N-terminal ectoplasmic domain of TbeIF2K2 that may serve a regulatory function is conserved in the orthologs of other trypanosomatids. It shares 31 and 24% identity (45 and 38% similarity) with eIF2K2 from T. cruzi and L. major, respectively. The T. cruzi protein is very similar to TbeIF2K2, containing a signal sequence at its N terminus and a transmembrane region in the exact same position as in the T. brucei protein. For the Leishmania protein, although clearly similar to TbeIF2K2 within the regulatory region, there are several inserts relative to TbeIF2K2. In addition, the signal sequence starts at residue 40, which may not qualify it for directing the protein to the membrane. It will certainly be interesting to localize the eIF2K2 proteins in the cells of both T. cruzi and L. major since these parasites seem to present slightly distinct exo- and endocytic systems relative to the better characterized membrane network described for T. brucei. The putative regulatory N-terminal domain does not show any sequence similarity with PEK/PERK or with any other known or predicted protein, including those of other parasites such as Plasmodium, Toxoplasma, Trichomonas, and Giardia. These observations taken together indicate that this kinase evolved only in the trypanosomatid lineage, but within it, it has somewhat diverged, perhaps reflecting differences in life cycles and cell biology.

	ACKNOWLEDGMENTS

 
We thank Ronald Wek (University of Indiana) for helpful discussions.

This study was supported by grants from Fundação de Amparo 'a Pesquisa no Estado de São Paulo (FAPESP) to B.A.C. and to S.S. and by National Institutes of Health grants AI056866 and AI35739 to J.D.B. M.C.S.M. and T.C.L.J. were supported by doctoral fellowships, and N.N.H. and V.S.A. were supported by postdoctoral fellowships from FAPESP.

	FOOTNOTES

 
* Corresponding author. Mailing address: Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, São Paulo, SP 04023-062, Brazil. Phone: (55)(11) 5576-4537. Fax: (55)(11) 5572-4711. E-mail: bacastilho{at}unifesp.br

Published ahead of print on 14 September 2007.

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