Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins Juliette Moyersoen 1, *, Jungwoo Choe 2, *, Abhinav Kumar 3, †, Frank G. J. Voncken 4 , Wim G. J. Hol 2,3 and Paul A. M. Michels 1 1 Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite ´ Catholique de Louvain, Brussels, Belgium; 2 Department of Biochemistry, University of Washington, Seattle, USA; 3 Biomolecular Structure Center, Howard Hughes Medical Institute, University of Washington, Seattle, USA; 4 Zentrum fu ¨ r Molekulare Biologie der Universita ¨ t Heidelberg, Germany It has been shown previously in various organisms that the peroxin PEX14 is a component of a docking complex at the peroxisomal membrane, where it is involved in the import of matrix proteins into the organelle after their synthesis in the cytosol and recognition by a receptor. Here we present a characterization of the Trypanosoma brucei homologue of PEX14. It is shown that the protein is associated with gly- cosomes, the peroxisome-like organelles of trypanosomatids in which most glycolytic enzymes are compartmentalized. The N-terminal part of the protein binds specifically to TbPEX5, the cytosolic receptor for glycosomal matrix proteins with a peroxisome-targeting signal type 1 (PTS-1). TbPEX14 mRNA depletion by RNA interference results, in both bloodstream-form and procyclic, insect-stage T. brucei, in mislocalization of glycosomal proteins to the cytosol. The mislocalization was observed for different classes of matrix proteins: proteins with a C-terminal PTS-1, a N-terminal PTS-2 and a polypeptide internal I-PTS. The RNA inter- ference experiments also showed that TbPEX14 is essential for the survival of bloodstream-form and procyclic trypano- somes. These data indicate the protein’s great potential as a target for selective trypanocidal drugs. Keywords: trypanosome; glycosome biogenesis; PEX14; protein–protein interactions; RNA interference. All protists grouped in the order Kinetoplastida have a unique form of metabolic compartmentation: the majority of the glycolytic enzymes is present in organelles called glyco- somes [1,2]. This compartmentation has been studied in detail in the kinetoplastid Trypanosoma brucei, the causative agent of African sleeping sickness. When present in the mammalian bloodstream, this parasite is totally dependent on glycolysis for its ATP supply as it lacks a functional Krebs’ cycle and the mitochondrial system for oxidative phosphorylation. In contrast, the procyclic trypomastigote living in the midgut of the tsetse fly relies more on mitochondrial activity. Because of its importance for the mammalian infective stage, glycolysis has been indicated as a validated target for the design of drugs against these parasites. The unique manner in which the pathway is organized in these organisms, and the observation that the structure and kinetic properties of most of their glycolytic enzymes display many special features, may allow the development of trypanocidal drugs that act through blocking specifically glycolysis in the parasites without interfering with the metabolism of the host [3]. The glycosome is an organelle related to peroxisomes [2,4,5]. Peroxisomes are compartments of eukaryotic cells that may harbour different enzymatic systems in organisms belonging to distinct taxonomic groups, but all peroxisomes have in common a number of metabolic functions such as an involvement in the metabolism of peroxides, fatty acids and ether lipids [6]. These functions are also shared by kinetoplastid glycosomes, which however, have glycolysis as their most distinctive feature. Considerable evidence is available to indicate that the presence of intact glycosomes and the proper targeting of glycolytic enzymes to the organelles is essential for kinetoplastid parasites [7–10]. Therefore glycosome biogenesis may also be a good target for drug interference. At least 23 proteins, called peroxins (PEX), have been identified in yeasts and mammalian cells as factors involved in the biogenesis of peroxisomes (reviewed in [11–14]). Peroxisomal matrix proteins are synthesized on free ribo- somes in the cytosol, and are translocated across the peroxisomal membrane usually without any form of pro- cessing. The proteins to be imported are recognized by a cytosolic receptor, either PEX5 or PEX7, depending on their peroxisome-targeting signal, PTS-1 or PTS-2, respectively. PTS-1 is a C-terminal tripeptide -SKL or a variation thereof. Correspondence to P. A. M. Michels, ICP-TROP 74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium. Fax: + 32 27626853, Tel.: + 32 27647473, E-mail: michels@trop.ucl.ac.be Abbreviations: ALD, aldolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEX, peroxin; PTS, peroxisome-targeting signal; PYK, pyruvate kinase; RNAi, RNA interference; Tet, tetracycline; TIM, triosephosphate isomerase. *Note: These authors contributed equally to this study. Present address: Plexxikon, Berkeley, California, USA. Note: The novel nucleotide sequence data published here have been deposited in the EMBL-EBI/GenBank and DDBJ databases and are available under accession number AJ512212. (Received 20 December 2002, revised 7 March 2003, accepted 18 March 2003) Eur. J. Biochem. 270, 2059–2067 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03582.x PTS-2 is the motif -R/K-L/V/I-X 5 -H/Q-A/L- close to the N-terminus. The receptor with its cargo docks at the peroxisomal membrane to a protein complex comprising PEX14, PEX13 and (so far only identified in yeast) PEX17. Several other peroxins are involved in the subsequent steps of the import. The import of matrix proteins seems to involve a cascade of interactions between the cargo-loaded receptors and different components of the import machinery: docking, translocation, cargo release and receptor recycling. In addition, some proteins are directed to the peroxisomes by a polypeptide-internal sequence (I-PTS), but so far, no specific, common motif could be identified for such I-PTS. PEX14, a protein associated with the peroxisomal mem- brane, has been identified in various yeasts, mammalian cells and plants as a component of the docking complex and a point of convergence of the PEX5- and PEX7-dependent import pathways [15–17]. The N-terminal part of PEX14 interacts with the repeated motifs WXXXF/Y of PEX5 [18] and with the SH3 domain of PEX13 through a proline-rich motif [15,17]. Interaction of PEX14 with PEX7 has also been shown [15]. PEX14 behaves as an integral peroxisomal mem- brane protein in Hansenula polymorpha [19] and humans [17] and as a tightly bound peripheral peroxisomal membrane protein exposed to the cytosol in Saccharomyces cerevisiae [15]. However, no membrane-spanning domains in PEX14 have been unambiguously identified and it was observed in mammalian cells and S. cerevisiae that both the N-terminal and the C-terminal domain are at the cytosolic face of the peroxisomal membrane [15,16,20]. The prediction of a membrane-anchoring subdomain is therefore speculative. Several peroxins involved in glycosome biogenesis have been identified in the kinetoplastids Leishmania and Trypanosoma [8,21–23]. Previously, we have characterized the T. brucei PTS-1 receptor, TbPEX5, and its interaction with glycosomal matrix proteins [22]. We have now cloned also the gene encoding PEX14 of T. brucei. Very recently, Furuya et al. [24], too, reported the identification of trypanosome homologue of PEX14. They showed that decreasing the level of this peroxin in procyclic T. brucei, by RNA interference, compromises import of a glycosomal matrix protein and renders glucose toxic to the cells. In this paper, we describe a more detailed analysis of the role of PEX14 in glycosome biogenesis in both bloodstream-form and procyclic trypanosomes. Moreover, we present data on the interaction of TbPEX14 with TbPEX5. Experimental procedures Organisms and growth conditions Bloodstream and procyclic form of T. brucei 427 (cell line 449 [25]) were cultured and transfected as described before [25]. The lines were not cloned. These phleomycin-resistant parasites express the tet repressor from the chromosomally integrated plasmid pHD449 and are metabolically indistin- guishable from the wild type [25]. Construction of expression clones; production of recombinant proteins PEX14 and PEX5 A short potential PEX14 sequence was recognized in the database of the T. brucei (strain TREU927/4) genome project. The corresponding fragment was amplified on T. brucei strain 427 genomic DNA and used as a hybridi- zation probe to screen a genomic library prepared of this strain in phage kGEM11 (Promega) [26]. Each part of the gene was sequenced at least once in both directions, using a Beckman CEQ 2000 sequencer (Beckman Instruments, Inc.). In order to overexpress TbPEX14 in Escherichia coli, the gene was introduced in the expression vector pET28a (Novagen) that directs the production of the protein fused to a N-terminal sequence including a histidine tag. E. coli strain BL21(DE3) which has the T7 RNA polymerase gene under the control of the lacUV5 promoter [27]. Also a fragment containing the first 146 amino acids (PEX14-N) was expressed, without tag. To this end, the corresponding 5¢ part of the gene and the complete T. brucei PEX14 gene were amplified by PCR using two sets of oligonucleo- tides: sense 5¢-GA CCATGGCTTTGCTGCTGTCGGG-3¢; 5¢-GT GGATCCTAATCCCTCCAGTCC-3¢ and antisense 5¢-GA GGATCCATGTCTTTGCTGCTG-3¢;5¢-CGAA GCTTTCAAGCTGCCTCGC-3¢, respectively. The sense primers contain a NcoIorBamHI site (underlined) adjacent to a sequence corresponding to the 5¢ end of the PEX14 gene, and the antisense primers are complementary to a gene-internal region or the terminal coding region of the gene, followed by a BamHI or HindIII restriction site (underlined), respectively. PCR was performed using Taq DNA polymerase (TaKaRa biomedicals). The amplified fragments were purified and ligated in the pGEM-T Easy vector (Promega). After checking their sequence, the amplified fragments were liberated from the recombinant plasmid by digestion with NcoIandBamHI or BamHI and HindIII, respectively, and ligated in the expression vector pET28a (Novagen). E. coli BL21(DE3) was then trans- formed with the recombinant pET28a-TbPEX14-N or pET28a-TbPEX14 plasmid. Only this latter construct directs the production of a fusion protein bearing an N-terminal extension of 20 residues including a His 6 -tag. At a later stage, a point mutation was introduced in the vector pET28a-TbPEX14-N in order to fuse the vector-encoded histidine tag also to the N-terminus of the TbPEX14-N sequence. This plasmid, pET28a-TbPEX14-N,H, was also introduced in E. coli BL21(DE3) and used for bacterial expression of recombinant protein TbPEX14-N,H. Cells harbouring a recombinant plasmid were grown at 37 °C in 50 mL of Luria–Bertani medium supplemented with 30 lgÆmL )1 kanamycin. Isopropyl thio-b- D -galacto- side was added to a final concentration of 1 m M when the culture reached an D 600nm of approximately 0.5 to induce the expression of the protein and growth was continued for 3 h. Cells were collected by centrifugation (3000 g, 10 min, 4 °C) and resuspended in cell lysis buffer containing 50 m M sodium phosphate, pH 8, 100 m M NaCl and a protease inhibitor mixture (Roche). Cells were lysed by two passages through an SLM-Aminco French pressure cell (SLM Instruments Inc.) at 90 MPa. The lysate was centrifuged (12 000 g, 15 min, 4 °C) and TbPEX14-N,H or TbPEX14 was purified from the soluble cell fraction. Nucleic acids were removed by treatment with 250 units Benzonase (Merck) for 30 min at 37 °C and protamine sulfate (0.5 mgÆmL )1 ) followed by centrifugation for 10 min at 12 000 g. The His-tagged proteins were further purified by 2060 J. Moyersoen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 metal affinity chromatography (TALON resin, Clontech) followed by anion-exchange chromatography (column 20HQ) and gel filtration (Superdex 200) to at least 90% purity. Gel-retardation, gel filtration Purified TbPEX5 [22] and TbPEX14-N,H were taken up in 5 m M Tris/HCl, pH 7.0, 100 m M NaCl and 1 m M dithiothreitol and incubated for 30 min at room tempera- ture. The sample was loaded on a 4–20% polyacrylamide gel (Novagen) and run under nonreducing and nondena- turing conditions for 90 min at 140 V with a Tris-glycine buffer of pH 8.5. The gel was stained with a gelcode blue stain solution (Pierce). Gel-filtration chromatography was performed on Super- dex 200 HR 10/30 (Amersham Biosciences) with 25 m M Tris/HCl, pH 7.0, 100 m M NaCl and 1 m M dithiothreitol at a flow rate of 0.4 mLÆmin )1 at 4 °C. A total volume of 100 lL of sample was loaded on the column. Six runs were performed with TbPEX5 alone, TbPEX14-N,H alone and a mixture of TbPEX5 and TbPEX14-N,H at various molar ratios. Protein concentrations were determined by the Bradford method using bovine serum albumin as standard [28]. This method was also used in experiments involving subcellular fractionation (see below). Construction of trypanosome expression vectors; RNAi To induce RNA interference (RNAi) in trypanosomes by the production of a double-stranded RNA, a 800-bp fragment [PEX14-(2–269)] from the PEX14 open-reading frame was cloned in the sense orientation upstream of the pSP72 ÔstufferÕ fragment [29] and in the antisense orientation downstream of the stuffer. The PEX14-(2–269)-stuffer- PEX14-(2–269) was cloned into the polylinker downstream of the tetracycline (Tet) inducible PARP promoter of the trypanosomatid vector pHD677 (giving pRP14) [25]. For induction of double-stranded RNA, cells were cultured in the appropriate medium [25] containing 250 ngÆmL )1 of Tet for bloodstream-form and 5 lgÆmL )1 of Tet for procyclic-form cells. Cultures were diluted daily to 2 · 10 5 cellsÆmL )1 for bloodstream-form trypanosomes or 1 · 10 6 cellsÆmL )1 for procyclic trypanosomes. Cell densi- ties were determined using a cell counting grid (Merck) and growth curves were plotted as the product of the cell density and the total dilution vs. time. Subcellular fractionation by treatment of cells with digitonin Trypanosomes (10 8 ) were washed twice in a buffer contain- ing 25 m M Hepes, pH 7.4, 250 m M sucrose and 1 m M EDTA, and then resuspended in the same buffer. Aliquots of a suspension of intact cells were diluted to a protein concentration of 1 mgÆmL )1 in HBSS buffer (Invitrogen). Subsequently, variable amounts of digitonin were added to the different aliquots followed by an incubation of 4 min at 25 °C. The suspensions were centrifuged at 12 000 g for 2 min. Aliquots of supernatants were used for Western blotting with rabbit polyclonal antisera raised against T. brucei pyruvate kinase (PYK, antiserum used at a dilution of 1 : 100 000), glyceraldehyde-3-phosphate dehy- drogenase (GAPDH, at 1 : 150 000), aldolase (ALD, at 1 : 150 000) and triosephosphate isomerase (TIM, at 1 : 10 000). Blots were processed for antigen detection using a peroxidase-conjugated affinity-purified rabbit anti- serum (Rockland) followed by a chemiluminescence-based detection system (ECL, Amersham Biosciences). Immunofluorescence Untreated procyclic trypanosomes or treated for 3 days with Tet were fixed and stained as described [30] using rabbit polyclonal anti-T. brucei ALD, GAPDH, PEX14 and PEX11 antisera. A synthetic peptide of the N-terminal part of TbPEX14 (DGKSKPEVEH), coupled to keyhole limpet hemocyanin, was used to raise a polyclonal antiserum in rabbit. The specificity of the antiserum was checked using a Western blot prepared after SDS/PAGE with a lysate of E. coli cells expressing TbPEX14-N,H. The single band detected has the molecular mass of the PEX14 fragment and was absent from noninduced bacteria. The anti-ALD Ig were purified on Protein A beads, coupled with Alexa- Fluor TM 488 (Molecular Probes) following the manufac- turer’s instructions. Detection of GAPDH, PEX14 and PEX11 was performed with Cy3-linked secondary antibody (Amersham, Biosciences). The software used was OPEN- LAB TM (Improvision) and ADOBE Ò PHOTOSHOP . Results T. brucei PEX14 interacts specifically with T. brucei PEX5 We have cloned and sequenced the PEX14 homologue of T. brucei strain 427, as described in Experimental proce- dures. The gene codes for a polypeptide (TbPEX14) of 365 amino acids, with a calculated molecular mass of 39 806 Da and a predicted isoelectric point of 6.07. The sequence is nearly identical to that reported recently by Furuya et al. [24] for the PEX14 of another strain (EATRO 110). Only one substitution was observed: a valine at position 156 instead of the alanine reported in [24]. In yeast, human, and Arabidopsis, it has been shown that the N-terminal part of PEX14 is responsible for the interaction with PEX5 [16,31,32]. This N-terminal domain of the polypeptide of each of these organisms is followed by a stretch of hydrophobic amino acids that is presumably involved in the protein’s anchoring to the membrane. Also in PEX14 of T. brucei a series of hydrophobic residues is found at the corresponding region of the polypeptide (amino acids 148– 166, LVIGAGAAVIGGFAAFKAF). Because this stretch may affect the solubility of the protein, we preferred to use only the soluble N-terminal segment of the molecule for in vitro interaction studies between TbPEX14 and TbPEX5. To this end, the first 146 amino acids of TbPEX14 were overexpressed in E. coli as a fusion protein with a His-tag called TbPEX14-N,H and purified (see Experimental pro- cedures). TbPEX5 was overexpressed and purified as described by de Walque et al.[22]. Gel retardation assays under nondenaturing and non- reducing conditions were performed to ascertain the Ó FEBS 2003 Trypanosoma brucei glycosomal PEX14 (Eur. J. Biochem. 270) 2061 association of the proteins TbPEX5 and TbPEX14-N,H (Fig. 1). The proteins were mixed and incubated for 30 min before subjected to electrophoresis. In lanes 1 and 7 (Fig. 1A), TbPEX5 and TbPEX14-N,H alone were, respectively, loaded on the gel. From the second lane to the sixth, mixtures of TbPEX5 and TbPEX14-N,H are present, with their ratio increasing from 1 : 0.6 to 1 : 6.8. These lanes show a shifted band supposedly corresponding to the complex formed by the two proteins (band 3). As the molar ratio increases, more unbound TbPEX14-N,H is seen in the upper part of the gel (Fig. 1A, band 5). To confirm the composition of the various bands, the proteins were extracted from the native gel and analysed by SDS/PAGE (Fig. 1B). Indeed both proteins form a complex and run together as they are present in the same band 3. On the native gel, band 4 (lane 6) seems to represent some TbPEX5 which does not bind to TbPEX14-N,H even in the presence of excess of the latter. This could be explained by release of some TbPEX5 from the interacting complex during migration. However, when proteins from band 4 were extracted from the native gel and subjected to SDS/ PAGE, not only TbPEX5, but also some TbPEX14-N,H was located in the band. This may be due to diffusion of some complexed protein as the band migrates in the native gel. Band 5 contains the excess of TbPEX14-N,H. In a second experiment, the same two components were also mixed in different molar ratios and analysed by gel- filtration chromatography. The elution profiles of the protein mixture, TbPEX5 and TbPEX14-N,H, contained an early eluting species, which was not detectable when each protein was analysed individually. This high molecular mass complex consists of TbPEX5 and TbPEX14-N,H as con- firmed by SDS/PAGE analysis. SDS/PAGE analyses of corresponding peak fractions confirmed the presence of both proteins in the shifted peak whereas the unbound TbPEX5 or TbPEX14-N,H was found as the late peak in all elution profiles (Fig. 2). From the experiments presented in Figs 1 and 2, we conclude that the N-terminal part of TbPEX14 (TbPEX14-N,H) directly interacts with TbPEX5 in vitro. Destruction of PEX14 mRNA by RNA interference (RNAi) is lethal for trypanosomes The effect of suppression of TbPEX14’s expression on the parasite’s growth rate and glycolytic enzyme localization was studied. Double-stranded RNA corresponding to essential genes is deleterious for trypanosomes because it induces specifically the degradation of the mRNAs by a process called RNA interference (RNAi) [33]. A construct that leads to the production of double-stranded TbPEX14 RNA in T. brucei was prepared as described in Experimen- tal procedures. The linearized plasmid DNA was inserted into a transcriptionally silent region of the genome by homologous recombination. The production of the double- stranded RNA was induced by the addition of Tet and resulted, in bloodstream-form trypanosomes, in reduced growth within 24 h followed by death of parasites. In contrast, the effect on procyclic-form trypanosome growth was less acute. Indeed, a decrease in cell number could only be seen after 4 days of the Tet system’s induction (Fig. 3A). Whereas all cells of the procyclic culture were killed, this appeared not to be the case for the culture of bloodstream- form trypanosomes. This will be discussed below. Western blotting using lysates of bloodstream-form trypanosomes and a polyclonal antiserum raised in rabbits against a synthetic peptide (see Experimental procedures) showed that PEX14 is a low abundance protein. This observation is similar to that reported by Guerra-Giraldez et al. [34] for T. brucei PEX2. Attempts to confirm the decrease of the PEX14 protein as a result of RNAi remained inconclusive. On Northern blots made with RNA from procyclic trypanosomes, however, low levels of PEX14 mRNA could be detected, which decreased upon induction of RNAi, whereas the concentration of control mRNA (for the glycolytic enzyme enolase) remained unchanged (Fig. 3B). The effect of RNAi was more difficult to show for bloodstream-form T. brucei because the level of PEX14 mRNA in these cells was significantly lower (not shown). In order to determine the influence of the reduction of the expression of TbPEX14 on the import of glycosomal matrix proteins, the subcellular distribution of glycolytic enzymes was analysed. This was carried out by treating the cells with increasing concentrations of digitonin. This detergent forms insoluble complexes with sterols, thus permeabilizing the different cellular membranes in a selective manner. The plasma membrane, having a higher sterol content than the glycosomal membrane, will have its integrity affected at a lower concentration of digitonin than the glycosomal membrane [34–37]. Therefore, enzymes sequestered in glycosomes will only be released from the cells at higher concentrations of the detergent than enzymes present in the cytosol. The appearance of the enzymes outside the cells was assayed by Western blotting (Fig. 4). In procyclic-form trypanosomes, 0.05 mgÆ(mg protein) )1 affects only the plasma membrane, as indicated by the release of the cytosolic marker PYK from the cells (into the supernatant fraction), whereas glycosomal enzymes (GAPDH, ALD and TIM) remain in the cells (pellet fraction, not shown). In Fig. 1. Gel-retardation assay of TbPEX5 and TbPEX14 interaction, under nondenaturing and nonreducing conditions. (A) Purified His 6 - tagged TbPEX5 and a protein comprising the N-terminal part of TbPEX14 (TbPEX14-N,H) were loaded on the gel separately (first and last lane) or mixed together (lane 2–6). The molar ratios of TbPEX5/ TbPEX14-N,H run in the various lanes are indicated underneath the gel. (B) SDS/PAGE analysis of native gel bands. Band 1 corresponds to TbPEX5 (first lane), band 2 to TbPEX14-N,H (last lane), band 3 to the complex formed by the two proteins (lane 3), and bands 4 and 5 correspond to noncomplexed TbPEX5 and TbPEX14-N,H, respect- ively (lane 4 and 5). 2062 J. Moyersoen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 2. Gel-filtration chromatography of TbPEX5 and Tb PEX14 interaction. (A) Purified His 6 -tagged TbPEX5 and TbPEX14-N,H were loaded separately (first and last profile) or mixed together in various molar ratios: 1 : 0.6, 1 : 0.9, 1 : 1.7, 1 : 3.4 (from bottom to top). Absorbance, OD, of the column eluate was monitored at 280 nm. The peaks at an elution volume of 12 mL and 15 mL correspond to TbPEX5 and TbPEX14-N,H, respectively. (B) SDS/PAGE analysis of the different peak fractions. The numbers at the top of the gel correspond to the different peaks of the elution profile. Fig. 3. Effect of PEX14-specific RNAi on the growth of bloodstream-form (BF) or procyclic-form (PF) T. brucei. (A) The growth profiles of cells transfected with the plasmid producing double-stranded RNA of TbPEX14 were determined by growing the cells in the absence (j)orinthe presence of Tet (r) (250 ngÆmL )1 for BF and 5 lgÆmL )1 for PF). Shown are cumulative cell numbers (determined as described in Experimental procedures). (B) Northern analysis of total RNA from uninduced (–Tet) and induced for 24 h (+Tet) procyclic-form trypanosomes. Each lane contains 10 lgtotalRNA. Ó FEBS 2003 Trypanosoma brucei glycosomal PEX14 (Eur. J. Biochem. 270) 2063 contrast, the glycosomal enzymes were only released at a higher concentration of digitonin, necessary to permeabilize the glycosomal membrane. In trypanosomes grown for 3 days with Tet, the subcellular distribution of enzymes was clearly affected: part of the GAPDH (a PTS-1 protein), ALD (a PTS-2 protein) and TIM (an I-PTS protein) is already released from the cells at concentrations of digitonin that leave the glycosomal membrane intact. A similar, partially cytosolic localization of these three enzymes was observed when bloodstream-form cells were treated for 24 h with Tet (data not shown). The partial mislocalization of all three enzymes in these cells clearly shows the involvement of PEX14 in the targeting of PTS-1, PTS-2 and I-PTS proteins to the glycosome. To further analyse the effect of the destruction of PEX14 mRNA on the parasite, we performed immunofluorescence experiments (Fig. 5). First we confirmed the glycosomal localization of TbPEX14. Indeed, trypanosomes labelled with anti-TbPEX14 presented a punctuate pattern and a colocalization of the protein with the glycosomal enzyme ALD in wild-type cells. We next examined the distribution of glycosomal matrix enzymes and a glycosomal membrane marker, PEX11 (Fig. 5B,C). In cells that were not treated with Tet, ALD and GAPDH, and ALD and PEX11, respectively, colocalized and showed a punctuate fluores- cence pattern corresponding to a glycosomal localization. However, many cells treated for 3 days with Tet were already severely affected as concluded from their round shape and disintegration of the nucleus (data not shown). Cells with apparently normal morphology were analysed. In the first +Tet panel (Fig. 5B), both ALD and GAPDH show a clear cytoplasmic localization. In the second panel, cells at an intermediate stage are shown, where ALD is still present in glycosomes while most of the GAPDH is present in the cytoplasm. In Fig. 5C, the nontreated trypanosomes have the same punctuate pattern as the wild-type cell in Fig. 5A. Together, these data indicate that TbPEX14 depletion does prevent proper targeting of glycosomal matrix enzymes. Surprisingly, the Tet-treated cells showed a partial cytoplasmic localization of PEX11 similar to that of ALD. Discussion In peroxisomal matrix protein import, PEX14 seems to be involved in the first event occurring at the peroxisomal membrane. The cytosolic receptors, PEX5 and PEX7, Fig. 4. Subcellular fractionation by treatment of cells with digitonin. Intact procyclic-form T. brucei cells were incubated for 4 min at con- centrations of digitonin as indicated. The release of PYK, ALD, GAPDH and TIM from the cells was assayed after centrifugation of the treated cell suspensions and the preparation of a Western blot of the resulting supernatants. The subcellular distribution of PYK, ALD, GAPDH and TIM is shown for procyclic-form cells grown without (–Tet) or with Tet for 3 days (+Tet). Fig. 5. Effects of PEX14 depletion on the distribution of glycosomal proteins of T. brucei, as determined by immunofluorescence. (A) Sub- cellular localization of TbPEX14 in wild type (WT) procyclic trypan- osomes. ALD is shown in green and PEX14 in red. (B) Effects of TbPEX14 depletion on matrix protein import. ALD is shown in green, and GAPDH in red. (C) Effects of TbPEX14 depletion on glycosomal matrix and membrane proteins in procyclic trypanosomes. ALD is shown in green, and PEX11 in red. In panels B and C, cells were grown in the presence (+Tet) or the absence (–Tet) of 5 lgÆmL )1 Tet for 3 days. Control experiments using only one antibody at a time and looking in two different channels have been performed to eliminate possible artefacts (not shown). 2064 J. Moyersoen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 loaded with their PTS-1 or PTS-2 proteins, respectively, dock at the peroxisomal membrane through PEX14. In this respect, some studies showed that the N-terminal part of PEX14 interacts with pentapeptide motifs, WXXXF/Y, present in several copies in the N-terminal domain of all PEX5s [22,32,38]. In this paper, in vitro experiments showed that also in T. brucei the N-terminal part of TbPEX14 (amino acids 1–146) interacts specifically with TbPEX5. This result is consistent with a role of PEX14 in glycosomal matrix protein import. While this manuscript was in preparation, Jardim et al. [39] reported the cloning and characterization of the Leishmania donovani PEX14 homologue. The leishmania PEX14 was shown to be tightly associated with the glycosomal membrane. Moreover, similar to our work, these authors showed by an in vitro analysis that the LdPEX14 binds to the previously characterized LdPEX5 [39] with a binding constant K d of 2.75 l M , and that the amino-terminal domains of the two peroxins were respon- sible for this interaction. The role of TbPEX14 in glycosome biogenesis received further confirmation by analysing the subcellular distribu- tion of the glycosomal enzymes GAPDH, ALD and TIM upon reduction of the PEX14 mRNA level. RNAi treatment of T. brucei resulted in the release of part of the glycosomal proteins from the cell at lower concentrations of digitonin than in untreated cells (Fig. 4). We interpret this observation as the retainment of newly synthesized proteins in the cytosol due to the inability to import matrix proteins by the fraction of growing and dividing glycosomes. The miscompartmentation of PTS-1 and PTS-2 proteins (GAP- DH and ALD, respectively) observed in both the procyclic and bloodstream-form trypanosomes when PEX14 expres- sion is affected, strongly suggests that also in T. brucei, this peroxin is the point of convergence of both matrix proteins import pathways. Highly interestingly, also TIM, which has no PTS-1 or PTS-2, is mislocalized in these cells. In trypanosomes transfected with different parts of the TIM gene fused to the gene of a reporter protein, the import signal was located in a 22-residue peptide in the center of the primary structure of TIM (S. de Walque & P. A. M. Michels, unpublished data). Moreover, we have not been able to show an interaction between T. brucei TIM and PEX5 in vitro under conditions that allowed PEX5 to interact specifically with a PTS-1 protein [22]. To our knowledge, no evidence for the involvement of PEX14 in I-PTS protein import has been described so far. The mechanism of I-PTS protein import in the peroxisomes is, at present, not known. We propose that I-PTS proteins such as TbTIM enter glycosomes by Ôpiggy backingÕ, involving the formation of a heteromeric complex with a PTS-1 or PTS-2 protein (see also [40,41]). Indeed, our observation of an altered TIM compartmentation in RNAi-treated cells is in line with such a mechanism. This hypothesis is currently under investigation in our laboratory. Surprisingly, also PEX11, a glycosomal membrane protein [8], was partially shifted to the cytosol. At present, it is not clear yet if this should be interpreted as an involvement of PEX14 in the insertion of PEX11 into the membrane. Alternatively, it could be imagined that reduction of (functional) glycosomes due to PEX14 depletion results in aberrant distribution of PEX11(andTIM)inthecell. The study of the importance of TbPEX14 for the parasite has shown that the suppression of peroxin expression by RNAileadstoareductioninthegrowthrateofT. brucei, both of the bloodstream and the procyclic form, followed by death of the cells. Therefore, TbPEX14 appears to be an essential protein for T. brucei. However, the reduction of growth rate appears in the bloodstream-form cells after already 24 h whereas the effect on growth of the procyclic form is seen only after 4 days of treatment. Indeed, bloodstream-form trypanosomes are entirely dependent on glycolysis and glycosomes for their ATP supply. From a computer simulation of the metabolism, it was concluded that an intact glycosomal membrane and proper enzyme compartmentation are of vital importance for these cells to compensate for a lack of activity regulation of hexokinase and phosphofructokinase by feedback loops [9]. In contrast, procyclic cells have a highly branched metabolic network by which the harmful effects of the unregulated kinases may be alleviated. In addition, the partially cytosolically located pentose-phosphate pathway in procyclics [42,43] may pro- vide an extra-glycosomal shunt for glucose catabolism that partially overcomes the problems caused by the miscom- partmentation of glycolytic enzymes. Thus a mistargeting of glycosomal enzymes could result in a more severe pheno- type in bloodstream-form trypanosomes than in procyclics. Similar differences between bloodstream-form and procyclic T. brucei have been observed when PEX2 was targeted by RNAi [34]. Also surprising was the observation that the culture of bloodstream-form trypanosomes, in contrast to that of procyclic cells, was not entirely killed within 4 days after RNAi induction. This could be attributed to the develop- ment of cells that became nonresponsive to RNAi. In three experiments we observed that the number of cells increased again after 4 days (see Fig. 3A). Indeed, the spontaneous development of such RNAi revertants has been observed by usandothersalsoinothercasesandappearstooccurmore frequently in bloodstream-form trypanosomes than in procyclic cell lines [44]. The effect may be due to either a rearrangement of the Tet repressor gene [45] or to deletion of the target gene insert [44]. Furuya et al. [24], too, reported recently the cloning and sequencing of the gene and confirmed the glycosomal localization of the encoded protein. These authors demon- strated that disruption of PEX14 function by RNAi resulted in death of trypanosomes of both bloodstream and procyclic-form cultures. Interestingly, removal of simple sugars from the medium allowed the procyclic trypano- somes to survive. We interpret these data as additional support for our hypothesis that proper compartmentation is particularly important during growth on sugars to prevent the unrestrained accumulation of sugar-phosphates that would otherwise result from the lack of regulation of sugar kinases, as mentioned above [9]. During growth on other carbon sources such as amino acids, trypanosomes do not rely on glycosomes for catabolism and, consequently, lesser harm may occur to the cell. Glycolysis in bloodstream-form trypanosomes is consid- ered as a valid and highly appropriate target for the design of trypanocidal drugs, because this process is of vital importance for the energy supply of the parasites and the sequestering of their glycolytic pathway into glycosomes is Ó FEBS 2003 Trypanosoma brucei glycosomal PEX14 (Eur. J. Biochem. 270) 2065 different from that in the host where it is a cytosolic process. This compartmentation endows the parasite enzymes with unique structural and kinetic features [3]. It has now also been shown that the compartmentation itself is essential to the trypanosomes [8,9,34] (this paper). This vital importance of proper compartmentation and intact organelles together with the very low level of conservation of peroxins provide additional promising drug targets: specific compounds may be designed that, in a selective manner, would interfere effectively with glycosome biogenesis, without affecting peroxisome biogenesis in the cells of the human host. The amino-acid sequence of T. brucei PEX14hasonly31% identity with that of its human homologue, and both we and Furuya et al. [24] showed that this peroxin is an essential protein for both bloodstream and procyclic-form trypano- somes. Therefore, PEX14 is a new, potentially good target for drugs that would act by disrupting the specific inter- actions between TbPEX14 and other peroxins, e.g. TbPEX5 or other T. brucei peroxins still to be identified and characterized (e.g. PEX13, PEX7). Acknowledgements This research was supported by grants from the Belgian Fonds de la Recherche Scientifique Me ´ dicale (FRSM) and the Interuniversity Attraction Poles – Federal Office for Scientific, Technical and Cultural Affairs. Juliette Moyersoen is a recipient of a scholarship from the Fonds a ` la Recherche dans l’Industrie et l’Agriculture (Belgium). We thank Dr Barbara Bakker (Amsterdam, the Netherlands) for stimula- ting discussions and critical reading of the manuscript. We also thank Dr Julius Lukes (Ceske Budejovice, Czech Republic) for raising an antiserum against PEX14. References 1. Opperdoes, F.R. (1987) Compartmentation of carbohydrate metabolism in trypanosomes. Annu. Rev. Microbiol. 41, 127–151. 2. Michels, P.A.M., Hannaert, V. & Bringaud, F. (2000) Metabolic aspects of glycosomes in Trypanosomatidae – New data and views. Parasitol. Today 16, 482–489. 3. Verlinde, C.L.M., Hannaert, V., Blonski, C., Willson, M., Pe ´ rie ´ , J.J., Fothergill-Gilmore, L.A., Opperdoes, F.R., Gelb, M.H., Hol, W.G.J. & Michels, P.A.M. (2001) Glycolysis as a target for the design of new anti-trypanosome drugs. Drug Resist. Updat. 4, 50–65. 4. Borst, P. (1989) Peroxisome biogenesis revisited. Biochim. Biophys. Acta 1008, 1–13. 5. Parsons, M., Furuya, T., Pal, S. & Kessler, P. (2001) Biogenesis andfunctionofperoxisomesandglycosomes.Mol. Biochem. Parasitol. 115, 19–28. 6. Van den Bosch, H., Schutgens, R.B.H., Wanders, R.J.A. & Tager, J.M. (1992) Biochemistry of peroxisomes. Annu. Rev. Biochem. 61, 157–197. 7. Blattner, J., Helfert, S., Michels, P.A.M. & Clayton, C. (1998) Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism. Proc. NatlAcad.Sci.USA95, 11596–11600. 8. Lorenz, P., Maier, A.G., Baumgart, E., Erdmann, R. & Clayton, C. (1998) Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p. EMBO J. 17, 3542– 3555. 9. Bakker, B.M., Mensonides, F.I.C., Teusink, B., Van Hoek, P., Michels, P.A.M. & Westerhoff, H.V. (2000) Compartmentation protects trypanosomes from the dangerous design of glycolysis. Proc.NatlAcad.Sci.USA97, 2087–2092. 10. Helfert, S., Este ´ vez, A., Bakker, B., Michels, P.A.M. & Clayton, C.E. (2001) Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei. Biochem. J. 357, 117–125. 11. Subramani, S., Koller, A. & Snyder, W.B. (2000) Import of per- oxisomal matrix and membrane proteins. Annu. Rev. Biochem. 69, 399–418. 12. Sacksteder, K.A. & Gould, S.J. (2000) The genetics of peroxisome biogenesis. Annu. Rev. Genet. 34, 623–652. 13. Terlecky, S.R. & Fransen, M. (2000) How peroxisomes arise. Traffic 1, 465–473. 14. Purdue, P.E. & Lazarow, P.B. (2001) Peroxisome biogenesis. Annu. Rev. Cell. Dev. Biol. 17, 701–752. 15. Albertini, M., Rehling, P., Erdmann, R., Girzalsky, W., Kiel, J.A.K.W., Veenhuis, M. & Kunau, W.H. (1997) Pex14p, a per- oxisomal membrane protein binding both receptors of the two PTS-dependent import pathways. EMBO J. 89, 83–92. 16. Brocard, C., Lametschwandtner, G., Koudelka, R. & Hartig, A. (1997) Pex14p is a member of the protein linkage map of Pex5p. EMBO J. 16, 5491–5500. 17. Will, G.K., Soukupova, M., Hong, X., Erdmann, K.S., Kiel, J.A.K.W., Dodt, G., Kunau, W.H. & Erdmann, R. (1999) Iden- tification and characterization of the human orthologue of yeast Pex14p. Mol. Cell. Biol. 19, 2265–2277. 18. Schliebs, W., Saidowsky, J., Agianian, B., Dodt, G., Herberg, F.W. & Kunau, W.H. (1999) Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14. J. Biol. Chem. 274, 5666–5673. 19. Komori, M., Kiel, J.A.K.W. & Veenhuis, M. (1999) The peroxi- somal membrane protein Pex14p of Hansenula polymorpha is phosphorylated in vivo. FEBS Lett. 457, 397–399. 20. Shimizu, N., Itoh, R., Hirono, Y., Otera, H., Ghaedi, K., Tateishi, K., Tamura, S., Okumoto, K., Harano, T., Mukai, S. & Fujiki, Y. (1999) The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis. J. Biol. Chem. 274, 12593–12604. 21. Flaspohler, J.A., Rickoll, W.L., Beverley, S.M. & Parsons, M. (1997) Functional identification of a Leishmania gene related to the peroxin 2 gene reveals common ancestry of glycosomes and peroxisomes. Mol. Cell. Biol. 17, 1093–1101. 22. de Walque, S., Kiel, J.A.K.W., Veenhuis, M., Opperdoes, F.R. & Michels, P.A.M. (1999) Cloning and analysis of the PTS-1 receptor in Trypanosoma brucei. Mol. Biochem. Parasitol. 104, 107–119. 23. Jardim, A., Liu, W., Zheleznova, E. & Ullman, B. (2000) Peroxi- somal targeting signal-1 receptor protein PEX5 from Leishmania donovani – molecular, biochemical, and immunocytochemical characterization. J. Biol. Chem. 275, 13637–13644. 24. Furuya, T., Kessler, P., Jardim, A., Schnaufer, A., Crudder, C. & Parsons, M. (2002) Glucose is toxic to glycosome-deficient try- panosomes. Proc. Natl Acad. Sci. USA 99, 14177–14182. 25. Biebinger, S., Wirtz, L.E., Lorenz, P. & Clayton, C. (1997) Vectors for inducible expression of toxic gene products in blood- stream and procyclic Trypanosoma brucei. Mol. Biochem. Parasitol. 85, 99–112. 26. Michels, P.A.M., Marchand, M., Kohl, L., Allert, S., Wierenga, R.K. & Opperdoes, F.R. (1991) The cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in Trypanosoma brucei have a distant evolutionary relationship. Eur. J. Biochem. 198, 421–428. 27. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89. 2066 J. Moyersoen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 28. Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 29. Shi, N., Djikeng, A., Mark, T., Wirtz, E., Tschudi, C. & Ullu, E. (2000) Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA. RNA 6, 1069–1076. 30. Maier, A., Lorenz, P., Voncken, F. & Clayton, C. (2001) An essential dimeric membrane protein of trypanosome glycosomes. Mol. Microbiol. 39, 1443–1451. 31. Saidowsky, J., Dodt, G., Kirchberg, K., Wegner, A., Nastainczyk, W., Kunau, W.H. & Schliebs, W. (2001) The di-aromatic penta- peptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal mem- brane protein PEX14. J. Biol. Chem. 276, 34524–34529. 32. Nito, K., Hayashi, M. & Nishimura, M. (2002) Direct interaction and determination of binding domains among peroxisomal import factors in Arabidopsis thaliana. Plant Cell Physiol. 43, 355–366. 33. Ngoˆ , H., Tschudi, C., Gull, K. & Ullu, E. (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. NatlAcad.Sci.USA95, 14687–14692. 34. Guerra-Giraldez, C., Quijada, L. & Clayton, C.E. (2002) Com- partmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei. J. Cell Sci. 115, 2651–2658. 35. Visser, N. & Opperdoes, F.R. (1980) Glycolysis in Trypanosoma brucei. Eur. J. Biochem. 103, 623–632. 36. Wiemer, E.A.C., Hannaert, V., Van den IJssel, P.R.L.A., Van Roy, J., Opperdoes, F.R. & Michels, P.A.M. (1995) Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypa- noplasma borelli. J. Mol. Evol. 40, 443–454. 37. Concepcio ´ n, J.L., Adje ´ , C.A., Quin ˜ ones, W., Chevalier, N., Dubourdieu, M. & Michels, P.A.M. (2001) The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi. Mol. Biochem. Parasitol. 118, 111–121. 38. Fransen, M., Brees, C., Ghys, K., Amery, L., Mannaerts, G.P., Ladant, D. & Van Veldhoven, P.P. (2002) Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay. Mol. Cell. Proteomics 1, 243–252. 39. Jardim, A., Rager, N., Liu, W. & Ullman, B. (2002) Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani. Mole- cular, biochemical, and immunocytochemical characterization. Mol. Biochem. Parasitol. 124, 51–62. 40. Rachubinski, R.A. & Subramani, S. (1995) How proteins pene- trate peroxisomes. Cell 83, 525–528. 41. Elgersma, Y. & Tabak, H.F. (1996) Proteins involved in peroxi- some biogenesis and functioning. Biochim. Biophys. Acta 1286, 269–283. 42. Heise, N. & Opperdoes, F.R. (1999) Purification, localisation and characterisation of glucose-6-phosphate dehydrogenase of Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 21–32. 43. Duffieux, F., Van Roy, J., Michels, P.A.M. & Opperdoes, F.R. (2000) Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. J. Biol. Chem. 36, 27559–27565. 44. Chen, Y., Hung, C H., Burderer, T. & Lee, G S.M. (2002) Development of RNA interference revertants in Trypanosoma brucei cell lines generated with a double stranded RNA expression construct driven by two opposing promoters. Mol. Biochem. Parasitol. 126, 273–279. 45. Roper, J.R., Guther, M.L., Milne, K.G. & Ferguson, M.A. (2002) Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei. Proc. Natl Acad. Sci. USA 99, 5884–5889. Ó FEBS 2003 Trypanosoma brucei glycosomal PEX14 (Eur. J. Biochem. 270) 2067 . this observation as the retainment of newly synthesized proteins in the cytosol due to the inability to import matrix proteins by the fraction of growing and dividing. (not shown). In order to determine the in uence of the reduction of the expression of TbPEX14 on the import of glycosomal matrix proteins, the subcellular