Glutathione transferases kappa and kappa localize in peroxisomes and mitochondria, respectively, and are involved in lipid metabolism and respiration in Caenorhabditis elegans ´ Elise Petit1,2,3, Xavier Michelet4,5, Claudine Rauch1,2,3, Justine Bertrand-Michel6, Francois Terce6,7,8, ¸ Renaud Legouis4,5 and Fabrice Morel1,2,3 ´ Inserm U620, Universite de Rennes 1, France ´ EA-MDC, Universite de Rennes 1, France IFR 140, Rennes, France ´ ´ ´ CNRS Centre de Genetique Moleculaire – UPR2167, Gif-sur-Yvette, France ´ ´ Universite Paris-Sud Orsay, Universite Paris-6, France ´ ´ ´ IFR150, Institut Federatif de Recherche Bio-Medicale de Toulouse, Plateau technique de Lipidomique, France INSERM, U563, Toulouse, France ´ ´ ´ ´ Universite Toulouse III Paul Sabatier, Departement Lipoproteines et Mediateurs Lipidiques, IFR150, France Keywords Caenorhabditis elegans; fatty acids; glutathione transferase kappa; mitochondria; peroxisomes Correspondence ˆ F Morel, INSERM U522 ⁄ EA MDC, Hopital Pontchaillou, 35033 Rennes, France Fax: +33 299540137 Tel: +33 299543737 E-mail: fabrice.morel@inserm.fr ´ ´ R Legouis, Centre de Genetique ˆ ´ Moleculaire – UPR2167 CNRS Batiment 26, Avenue de la terrasse, 91198 Gif-sur-Yvette Cedex, France Tel: +33 169824374 Fax: +33 169824386 E-mail: legouis@cgm.cnrs-gif.fr (Received 28 May 2009, revised July 2009, accepted July 2009) doi:10.1111/j.1742-4658.2009.07200.x To elucidate the function of kappa class glutathione transferases (GSTs) in multicellular organisms, their expression and silencing were investigated in Caenorhabditis elegans In contrast with most vertebrates, which possess only one GST kappa gene, two distinct genes encoding GSTK-1 and GSTK-2 are present in the C elegans genome The amino acid sequences of GSTK-1 and GSTK-2 share around 30% similarity with the human hGSTK1 sequence and, like the human transferase, GSTK-1 contains a C-terminal peroxisomal targeting sequence gstk-1 and gstk-2 genes show distinct developmental and tissue expression patterns We show that GSTK-2 is localized in the mitochondria and expressed mainly in the pharynx, muscles and epidermis, whereas GSTK-1 is restricted to peroxisomes and expressed in the intestine, body wall muscles and epidermis In order to determine the potential role(s) of GST kappa genes in C elegans, specific silencing of the gstk-1 and gstk-2 genes was performed by an RNA interference approach Knockdown of gstk-1 or gstk-2 had no apparent effect on C elegans reproduction, development, locomotion or lifespan By contrast, when biological functions (oxygen consumption and lipid metabolism) related to peroxisomes and ⁄ or mitochondria were investigated, we observed a significant decrease in respiration rate and a lower concentration of the monounsaturated fatty acid cis-vaccenic acid (18:1x7) when worms were fed on bacteria expressing RNA interference targeting both gstk-1 and gstk-2 These results demonstrate that GST kappa, although not essential for the worm’s life, may be involved in energetic and lipid metabolism, two functions related to mitochondria and peroxisomes Abbreviations Dsba, protein disulfide isomerase A; FAME, fatty acid methyl ester; GFP, green fluorescent protein; GST, glutathione transferase; PTS1, peroxisomal targeting signal 1; RNAi, RNA interference 5030 FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS E Petit et al Glutathione transferase kappa in C elegans Introduction Glutathione transferase (GST) kappa is a 26.5 kDa protein that was initially isolated from the rat liver mitochondrial matrix and classified as a class theta GST [1] The determination of the three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1), complexed with glutathione (GSH), showed a different folding topology from that of the other GST classes, and revealed that the enzyme shows similarity with the protein disulfide bond isomerase, DsbA, from Escherichia coli and a bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in the naphthalene degradation pathway [2,3] Although class kappa GST showed an activity towards aryl halides, such as 1-chloro-2,4-dinitrobenzene, and can reduce cumene hydroperoxide and (S)-15-hydroperoxy-5,8,11,13-eicosatetraenoic acid [4], this activity remained quite low when compared with that of other soluble GSTs Interestingly, a recent study has shown that GST kappa might also possess a function independent of its glutathione conjugation activity in adipose tissue [5] Indeed, Liu et al [5] have identified GSTK1 as a key regulator for the multimerization of adiponectin, which is an adipocyte-derived hormone, in both human and rodent Tissue distribution, analysed by RT-qPCR, showed that the hGSTK1 gene is expressed in the 24 different human tissues examined [4] In the mouse, the mGSTK1 protein is present in large amounts in the liver, kidney, stomach and heart, and its association with liver and kidney mitochondria has been demonstrated by electron microscopy [6] GSTK1 transcript tissue expression is similar in the rat and in the mouse [7] With regard to subcellular localization, in contrast with soluble GSTs, which are mainly present in the cytosol, GST kappa is localized in peroxisomes and mitochondria [4] Although the process of GST kappa targeting to mitochondria is unclear, it has been reported to associate with the Hsp60 chaperone [3], and a possible cleavage site for a mitochondrial presequence exists at the N-terminus A peroxisomal targeting sequence (tripeptide ARL) has been identified in the C-terminus of the hGSTK1 subunit [4] The recent demonstration of GST kappa as a regulator of protein multimerization and its particular subcellular location have led to questions about its further role(s) and substrate(s) [5] Common peroxisomal and mitochondrial functions are related to lipid metabolism, including a- and b-oxidation of fatty acids that generate acetyl-CoA and different acyl-CoA intermediates [8,9] Thus, the presence of GSTK1 in both organelles suggests that it may be specifically involved in the b-oxidation of fatty acids, either through its catalytic activity, a certain transport function or interaction with other proteins Interestingly, its role in adiponectin regulation is also related to lipid and glucose metabolism The nematode Caenorhabditis elegans is a genetically well-characterized model organism [10] which presents several advantages: (a) small size; (b) rapid reproduction as a self-fertile hermaphrodite; (c) large number of offspring (250–300 progeny); (d) growth on a solid surface medium; and (e) transparent body allowing the observation of cells in mature and developing animals Furthermore, as about 60% of C elegans genes show similarity to human genes, and transient RNA interference (RNAi) allows specific gene silencing, this model organism represents a powerful tool for gene function analysis The aim of our study was to characterize GST kappa gene(s) and proteins in C elegans and, by means of RNAi, to investigate the effects of gene silencing on the nematode phenotype Our results showed that the C elegans genome contains two GST kappa genes encoding GSTK-1 and GSTK-2, which localize in peroxisomes and mitochondria, respectively Double inactivation by RNAi affects the worm’s metabolism through a reduction in its rate of respiration and modification of its lipid content Results The C elegans genome contains two GST kappa genes We have previously described a C elegans protein showing 33% homology with the human GST kappa, hGSTK1, amino acid sequence [4] Database analyses revealed the presence of two genes previously named ZK1320.1 and D2024.7 in the C elegans genome These two genes are located on chromosomes II (ZK1320.1) and IV (D2024.7) and have probably arisen by gene duplication Both genes are composed of three exons and two introns (Fig 1A), the nucleotide sequence at the splice junctions is consistent with the canonical GT–AG rule and the corresponding encoded amino acid sequences comprise 226 and 225 residues, respectively, and share 32% identity Orthologues of these genes are observed in other nematode species, including C briggsae, C remanei, C japonica, Ancylostoma ceylanicum, Heterorhabditis bacteriophora and Meloidogyne Three arguments strongly suggest FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS 5031 Glutathione transferase kappa in C elegans A 69 gstk-1 (zk1320.1) B AA position rGSTK1 GSTK-1 GSTK-2 462 51 150 182 151 gstk-2 (d2024.7) E Petit et al 380 599 50 469 100 150 200 C that C elegans ZK1320.1 and D2024.7 genes belong to the kappa class of GSTs and share a common ancestral gene with rat GSTK1 Firstly, there are conserved amino acids in the protein sequences of rat GSTK1 and C elegans ZK1320.1 and D2024.7 Secondly, we showed common exon–exon junctions in the translated amino acid sequences of the two C elegans genes and the rat GST kappa gene (Fig 1B) Finally, using the psortii program (http://psort.ims.u-tokyo.ac.jp), the presence of a C-terminal peroxisomal targeting signal (PTS1), composed of the three amino acids serine-lysine-leucine (SKL), was demonstrated in some, but not all, nematode species and rat GST kappa (tripeptide ARL) amino acid sequences (Fig 1C) Furthermore, psortii also predicted a mitochondrial presequence with a putative cleavage site after residue (MPNRK ⁄ VV) at the N-terminus of the GSTK-2 sequence For these reasons, ZK1320.1 and D2024.7 genes were renamed gstk-1 and gstk-2 5032 147 Fig Genomic structure and intron positions in C elegans gst kappa genes (A) Genomic structure of hGSTK1 Exons are represented as black boxes and introns are represented by lines; the numbers indicate the size in nucleotides The gene structure is drawn to scale (B) Intron positions in rat and C elegans amino acid sequences Filled and open triangles mark common and unique intron positions, respectively (C) Amino acid sequence alignment of rat GSTK1 with GST kappa of nematodes The aligned sequences are listed below, followed by the species’ names and accession numbers in parentheses rGSTK1 (Rattus norvegicus, UniProt: P24473), CelGSTK-1 (Caenorhabditis elegans, UniProt: Q09652), CbrGSTK-1 (Caenorhabditis briggsae, UniProt: A8XB52), CelGSTK-2 (Caenorhabditis elegans, UniProt: Q18973), CbrGSTK-2 (Caenorhabditis briggsae, UniProt: A8X1K2), CreGSTK-2 (Caenorhabditis remanei, WormBase: RP16274), CjaGSTK-1 (Caenorhabditis japonica, WormBase: JA07681), AcGSTK (Ancylostoma ceylanicum, GenBank: CB175111.1), MhGSTK (Meloidogyne hapla, GenBank: EX007447.1), HbGSTK (Heterorhabditis bacteriophora, GenBank: BM883827.1) *Residues involved in glutathione binding site #Residues involved in dimer interface GSTK-1 and GSTK-2 are localized in peroxisomes and mitochondria, respectively The analysis of GST kappa transcript levels has shown a ubiquitous expression in human [4] and mouse [6] tissues In order to determine the spatial and temporal expression patterns of gstk-1 and gstk-2 genes in C elegans, we constructed gfp::gstk-1 and gstk-2::gfp fusions under the control of approximately kb of the 5¢ regulatory gstk-1 and gstk-2 sequences, respectively In these reporter fusion proteins, green fluorescent protein (GFP) was inserted in frame immediately upstream or downstream of gstk-1 and gstk-2 sequences, respectively Transgenic strains were obtained by microinjection, and the localization of fusion proteins in animals was revealed by the examination of GFP fluorescence (Fig 2) The expression of the gfp::gstk-1 transgene was first detected in 100 cell embryos, as shown in Fig 2A FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS E Petit et al Glutathione transferase kappa in C elegans A A' E B F C G D E' H Fig Analysis of expression pattern of gstk-1 and gstk-2 in embryo and adult C elegans Projection of confocal images of GFP::GSTK-1 (A–D) and GSTK-2::GFP (E–H) at different developmental stages (A) GFP::GSTK-1 is first detected at mid-embryogenesis in the primordium of the intestine (white arrow) and in the epidermis (arrowheads) A¢ is the corresponding Nomarski picture (B) During larval development, GFP::GSTK-1 is very strongly expressed with a vesicular localization in the intestine (arrows) (C) A weaker expression of GFP::GSTK-1 is present in the muscles (arrowheads) and the epidermis (arrows) (D) Faint diffuse expression is detected in the rectal gland cells (compare with intestinal signal indicated with an arrow) (E) GSTK-2::GFP is first detected in muscle quadrants (arrowheads) during morphogenesis of the embryo E¢ is the corresponding Nomarski picture (F) In larvae, a strong punctate staining is present in the pharynx (arrows) and the body wall muscles (arrowheads), and a weaker signal is observed in the intestine (G) In the pharynx, a very regular expression of GSTK2::GFP in myo-epithelial cells (arrow) is characteristic of a mitochondrial localization (H) In body wall muscle cells, GSTK-2::GFP is detected as a tubular network with stronger punctata (arrows) typical of the mitochondrial system Scale bar, 10 lm Expression was increased during morphogenesis of the embryo Fluorescence was observed as strong punctate staining in intestinal cells and in the epidermis The number and intensity of fluorescent structures increased strongly in the intestine during larval development (Fig 2D), whereas the epidermal fluorescent punctata weakened (Fig 2B) In addition, a diffuse localization of GFP::GSTK-1 was observed in the body wall muscles (Fig 2C) and in the rectal gland cells in larvae and adults (Fig 2D) The presence of a peroxisomal targeting signal in its C-terminus (Fig 1) and the punctate localization pattern suggest that GSTK-1 is a peroxisomal protein To confirm the peroxisomal localization, we masked PTS1 by fusing the GFP at the C-terminus of GSTK-1 Transgenic worms for GSTK-1::GFP only presented diffuse staining (data not shown), further supporting a peroxisomal localization of GSTK-1 In the GSTK-2::GFP transgenic strain, fluorescence was first detected during the second half of embryogenesis (Fig 2E) A strong signal was detected as punctate staining in the pharynx (Fig 2G) and the body wall muscles (Fig 2F), and a weaker signal was also observed in the intestine In muscle cells, the strong GSTK-2::GFP punctata were part of a tubular network which was weakly fluorescent (Fig 2H) Interestingly, similar staining has been observed previously by the expression of GFP fused to specific subcellular targeting sequences [11], strongly suggesting the presence of GSTK-2 in the mitochondria To confirm this mitochondrial localization, we stained both GFP::GSTK-1- and GSTK-2::GFP-expressing worms with MitoTracker Red (Fig 3) Although GFP:: GSTK-1 did not show any colocalization with MitoTracker Red staining (Fig 3A¢¢), GSTK-2::GFP fully colocalized with the mitochondrial dye (Fig 3B¢¢) Together, these data indicate that GSTK-1 and GSTK-2 have a peroxisomal and mitochondrial localization, respectively Impairment of oxygen consumption and lipid content in gstk-1 and gstk-2 double-knockdown worms Post-transcriptional gene silencing of specific genes by RNAi is a well-established method in C elegans [12] FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS 5033 Glutathione transferase kappa in C elegans E Petit et al A A' A'' B B' B'' Fig GSTK-2, but not GSTK-1, localizes in mitochondria Single confocal images of GFP::GSTK-1 (A) and GSTK-2::GFP (B) in adult animals GSTK-2::GFP fully colocalizes with the mitochondrial-specific marker MitoTracker (A¢), whereas GFP::GSTK-1 is not localized in the mitochondrial network (B¢) Scale bar, 10 lm 5034 100 Control 90 gstk-1 (RNAi) 80 gstk-2 (RNAi) 70 Viability (%) In our study, C elegans (strain N2) was fed with bacteria producing dsRNA of the gstk-1 and ⁄ or gstk-2 coding regions In each experiment, four feeding conditions were defined: one group of worms was fed with control bacteria containing the empty plasmid pL4440, one with bacteria expressing gstk-1(RNAi), another with bacteria expressing gstk-2(RNAi) and one with a mix (1 : 1) of both RNAi-expressing bacteria To test the efficiency of RNAi, fluorescence levels in RNAitreated GSTK::GFP transgenic worms were compared with the levels observed in control worms Figure S1 (see Supporting information) shows that RNAi directed against either gstk-1 (Fig S1A) or gstk-2 (Fig S1E) was efficient, with the exception of the pharynx in gstk-2::gfp transgenic worms, where silencing was incomplete (Fig S1E,F) It is noteworthy that gstk-2(RNAi) had no effect on the transgenic strain expressing GFP::GSTK-1 (Fig S1B), and GSTK2::GFP expression remained unchanged in transgenic animals fed with bacteria producing gstk-1 dsRNA (Fig S1D), indicating the specificity of these RNAi forms and the absence of compensatory adaptation [i.e upregulation of gstk-1 in gstk-2(RNAi) worms] RNAi silencing of gstk-1 or gstk-2 had no apparent effect on C elegans reproduction or development (data not shown) This absence of obvious phenotype has been reported previously in wide RNAi screens (http:// gstk-1/k-2 (RNAi) 60 50 40 30 20 10 0 10 15 Days 20 25 30 Fig gstk-1(RNAi) and gstk-2(RNAi) not affect the C elegans lifespan Effects of RNAi-mediated knockdown of gstk-1 and ⁄ or gstk-2 on the lifespan in wild-type worms Worms were fed either with control bacteria not expressing any dsRNA or bacteria expressing dsRNA that targets gstk-1, gstk-2 or both gstk-1 and gstk-2 Nematode survival was analysed by the Kaplan–Meier method using Graphpad Prism The same software was used to test the equality of survival with the log-rank (Wilcoxon) test Each experimental condition was tested in triplicate www.wormbase.org) We also found that these RNAi forms did not affect the C elegans lifespan (Fig 4) The first animal died after days and all animals were FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS O2 absorbed (nmol·min–1·1000–1 worms) E Petit et al Glutathione transferase kappa in C elegans * 20 15 10 Control gstk-1 (RNAi) gstk-1/k-2 (RNAi) gstk-2 (RNAi) RNAi Strain Fig gstk-1 ⁄ gstk-2(RNAi) animals present an altered respiratory rate Oxygen consumption was assessed in the fourth larval stage of animals fed either with control bacteria not expressing any dsRNA or bacteria expressing dsRNA that targets gstk-1, gstk-2 or both gstk-1 and gstk-2 Results are the mean of six values ± standard deviation, and are expressed as nmoles of O2 per minute per 1000 worms Student’s t-test was applied for statistical studies between RNAi-fed worms and control worms (*P £ 0.05) dead after 32 days The mean lifespan for wild-type, gstk-1(RNAi), gstk-2(RNAi) and gstk-1+gstk-2 (RNAi) were 14, 14, 13 and 13 days, respectively These results indicate that gst kappa genes are not essential for the survival of C elegans As our expression data for GSTK-1 and GSTK-2 supported peroxisomal and mitochondrial localizations, we further investigated cellular functions, such as lipid metabolism and oxygen consumption, which are closely related to these two organelles The control worms showed an oxygen consumption of 12.2 nmolỈ min)1 per 1000 worms Interestingly, a significant decrease in oxygen consumption of about 40% (7.5 nmolỈmin)1 per 1000 worms) was observed when worms were fed on bacteria expressing dsRNAs targeting both gstk-1 and gstk-2 (Fig 5) However, no significant decrease in oxygen consumption was observed in worms fed with gstk-1(RNAi) or gstk2(RNAi) alone compared with the control condition Thereafter, in order to investigate the effect of gstk-1 and ⁄ or gstk-2 silencing on lipid metabolism, we measured the following lipid fractions: phospholipids, diglycerides, triglycerides, free or esterified cholesterol, free fatty acids and total fatty acids Although most lipid concentrations were unchanged (Tables S1–S4, see Supporting information) between wild-type and gstk-1(RNAi), gstk-2(RNAi) and gstk-1 ⁄ gstk-2(RNAi) worms, a difference was observed for cis-vaccenic acid (18:1x7) for the fatty acid methyl ester (FAME) fraction, which was decreased significantly in worms depleted for both gstk-1 and gstk-2 (Fig 6) These data strongly suggest that GSTK-1 and GSTK-2 have overlapping functions, as oxygen consumption and cis-vaccenic acid levels were unchanged in gstk1(RNAi) and gstk-2(RNAi) worms and decreased only in double-knockdown animals Discussion In this study, we investigated the localization and potential role(s) of GST kappa in C elegans In contrast with most vertebrates, the C elegans genome contains two GST kappa genes, gstk-1 and gstk-2 The two genes are located on different chromosomes and contain three exons Interestingly, orthologues of C elegans gstk-1 and gstk-2 genes are also found in Control gstk-1(RNAi) gstk-2(RNAi) gstk-1/k-2(RNAi) * 18 16 Fig gstk-1 ⁄ gstk-2(RNAi) animals display an abnormal FAME composition Simplified FAME composition of control and gstk-1, gstk-2 and double gstk-1(RNAi) and gstk-2(RNAi)-treated animals (see also Tables S1–S4) Depletion of both gstk-1 and gstk-2 leads to a decrease in the 18:1w7 fatty acid, but does not affect other lipids The results are the mean of three experiments ± standard deviation Student’s t-test was applied for statistical studies between RNAi-fed worms and control worms (*P £ 0.05) % of total fatty acids 14 12 10 16:0 18:0 16:1w7 FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS 18:1w9 18:1w7 18:2w6 20:5w3 17 Cyclo Fatty acid mono esters 5035 Glutathione transferase kappa in C elegans E Petit et al the nematode species C briggsae and C remanei, indicating that a gene duplication took place before the speciation of these three Caenorhabditis species Sequence conservation between the GSTs of C elegans gstk-1 ⁄ and those of other nematode species (C briggsae, C remanei, C japonica, Ancylostoma ceylanicum, Heterorhabditis bacteriophora and Meloidogyne hapla), as well as with rat GSTK1, is also observed, the most highly conserved residues being those that contribute to the glutathione-binding site and dimerization of the protein Interestingly, structure prediction and molecular modelling studies have shown that, despite the low sequence similarity (30%), rGSTK1 and hGSTK1 structures are recognized as the closest structural homologues of C elegans GSTK-1 and GSTK-2 (data not shown) Together, these observations suggest that C elegans GSTK-1 and GSTK-2 might have similar activities to their mammalian orthologues [3,4,7], and may contribute, at least in part, to detoxification processes Moreover, a tripeptide sequence (SKL), known as PTS1, is present at the C-terminal end of the C elegans GSTK-1 protein PTS1 is involved in protein import into peroxisomes, and the importance of this signal for peroxisome targeting in C elegans has been shown previously by Motley et al [13] Interestingly, GFP fused to the N-terminus of GSTK-1 was found to localize in punctate bodies of C elegans cells in several tissues, strongly suggesting a peroxisomal localization By contrast, colabelling with MitoTracker Red showed that GSTK-2::GFP was present mainly in the mitochondria It is noteworthy that, in human cells, hGSTK1 is both peroxisomal and mitochondrial and contains a C-terminal PTS1 [4] This preserved intracellular localization of GST kappa of both nematodes and vertebrates, together with sequence conservation and intron positions in amino acid sequences, indicate that kappa class genes probably originated from a common ancestral gene which was present before the protostome ⁄ deuterostome split Interestingly, the presence of two duplicated paralogous genes, gstk-1 and gstk-2, in the C elegans genome allowed specialization of the subcellular localization for each gene The use of reporter fusion proteins allowed the study of tissue expression patterns of gst kappa genes Although both GFP::GSTK-1 and GSTK-2::GFP fusion proteins are observed in common tissues, such as the intestine, they also have a specific localization in other tissues, such as the epidermis or pharynx for GFP::GSTK-1 and GSTK-2::GFP, respectively In C elegans, the intestine and epidermis are at the interface between the organism and its environment Therefore, these tissues represent defence barriers 5036 against toxic agents, such as gut-derived oxidants or endogenously generated reactive oxygen species The intestine is also a highly metabolically active organ and represents the tissue in which most C elegans peroxisomes are found, as shown by immunostaining for catalase [14] and by electron microscopy [15] Interestingly, GSTK2::GFP is predominantly expressed in muscle cells (pharynx and body wall muscle) The expression of GST kappa genes in body wall muscle and pharynx might be related to the large number of mitochondria in these two tissues, which are associated with high energy consumption In order to gain further insight into the potential function(s) of GSTK-1 and GSTK-2, RNAi was used to knock down the expression of the two corresponding genes, either separately or simultaneously Knockdown of gstk-1 and ⁄ or gstk-2 had no effect on worm lifespan, locomotion or development, suggesting that GST kappa genes are not essential for the worm’s life Because, as in mammals, peroxisomes and mitochondria in C elegans play a key role in the production of reactive oxygen species and in lipid metabolism, including fatty acid b-oxidation [16], we investigated the potential role of gstk-1 and ⁄ or gstk-2 on the lipid composition of worms For this purpose, phospholipids, diglycerides, triglycerides, free and esterified cholesterol, and free and total fatty acid levels were measured in worms fed on gstk-1(RNAi) and ⁄ or gstk2(RNAi) With the exception of cis-vaccenic acid (18:1x7) from the FAME fraction, there was no modification of lipid composition between worms fed on the empty vector control RNAi and those fed on gstk-1(RNAi) and ⁄ or gstk-2(RNAi) It is noteworthy that the concentration of cis-vaccenic acid methyl ester was decreased only in double-knockdown (gstk-1 and gstk-2) worms Vaccenic acid is the most abundant fatty acid in phospholipids and triglycerides [17], and is elongated from palmitoleic acid (16:1x7) Another phenotypic feature of double-knockdown (gstk-1 and gstk-2) worms was the impairment of oxygen consumption It is also noteworthy that vaccenic acid synthesis and worm respiration are closely linked to peroxisomal and ⁄ or mitochondrial activities Interestingly, vaccenic acid is an important component of cardiolipin in different animal species [18], and this phospholipid plays a key role in mitochondrial function, particularly at the respiratory chain level [19] Although the link between decreased vaccenic acid levels and impairment in oxygen consumption merits further investigation, the presence of altered phenotypes only in double-knockdown worms indicates compensatory roles for GSTK-1 and GSTK-2 and suggests overlapping functions As peroxisomes and mitochondria FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS E Petit et al are metabolically linked, cooperate and cross-talk, especially in the b-oxidation of various fatty acids and in the maintenance of homeostasis in cellular reactive oxygen species [20,21], our results further strengthen the close relationship between these two organelles It is noteworthy that specific knockdown of either gstk-1 or gstk-2 was not accompanied by an upregulation of the paralogous gene Such compensatory responses have been demonstrated in Gst alpha (Gsta4) or Gst zeta (Gstz1) knockout mice, where the expression of other Gst classes and antioxidant enzymes was induced [22,23] By contrast, knockout of Gst pi1 ⁄ (Gstp1 ⁄ 2) did not lead to the upregulation of at least class alpha and mu transferases, as reported by Henderson et al [24] As the C elegans genome contains more than 50 genes encoding zeta, sigma, pi and omega class GSTs [25], further studies are needed to determine whether GSTs belonging to these classes or other genes are upregulated in gstk-1 and ⁄ or gstk-2 knockout worms With regard to the potential role of GST kappa, either direct or indirect, in lipid metabolism and respiration, it stills remain unclear One hypothesis might be that GST kappa plays a role in the folding of proteins involved in lipid synthesis or respiration Indeed, it has been demonstrated that GST kappa shares sequence and secondary structure homology with E coli protein disulfide bond isomerase (DsbA) and has the same general folding as DsbA The DsbA family is a subfamily of the thioredioxin family and catalyses disulfide bond formation during the folding of secreted proteins in bacterials [26] Recently, Liu et al [5] have shown that mouse and human GST kappa, renamed DsbA-L by these authors, are highly expressed in adipose tissue and interact with adiponectin Adiponectin is an adipokine specifically secreted from adipose tissue, which plays a key role in glucose and lipid metabolism in insulin-sensitive tissues [27] Overexpression of GST kappa promotes adiponectin multimerization by the formation of disulfide bonds between trimers [5] Although there is no adiponectin gene in the C elegans genome, GST kappa might have conserved such a role in the regulation of protein multimerization or interaction Certain proteins involved in lipid metabolism can exist as both monomers and dimers, for example the fatty acid synthase complex, and it has been demonstrated that some desaturases also form dimers [28,29] Similarly, several mitochondrial proteins form disulfide-linked multimeric complexes [30] Thus, a possible role of GST kappa might be to favour specific protein–protein interactions, and the absence of such interactions in double-knockdown (gstk-1 and gstk-2) worms may lead to lipid metabolism and respiration impairment Further investigation will Glutathione transferase kappa in C elegans be needed to confirm this hypothesis and to determine which proteins might be regulated by GST kappa In conclusion, this work has allowed the characterization of two GST kappa genes, gstk-1 and gstk-2, in the C elegans genome The products of these genes are differentially expressed in worm tissues and show distinct subcellular localizations, namely peroxisomal for GSTK-1 and mitochondrial for GSTK-2 Specific repression of each gene has no consequences on the worm phenotype By contrast, double-knockdown (gstk-1 and gstk-2) worms show decreased vaccenic acid levels and lower oxygen consumption when compared with wild-type worms Materials and methods Caenorhabditis elegans strains Caenorhabditis elegans cultures were grown and maintained at 20 °C using NGM agar plates supplemented with lgỈmL)1 of cholesterol [10] The wild-type reference strain Bristol N2 was used All experiments were performed at 20 °C Fluorescent-tagged protein constructs and the production of transgenic animals Reporter gene constructs were obtained by a PCR fusionbased approach [31] Genomic gstk-2 (D2024.7), with 1.8 kb immediately upstream of the start codon, was PCR amplified from wild-type genomic DNA using a TripleMasterPCR System (Eppendorf, Hamburg, Germany) This product was then coamplified with a 1.8 kb PCR fragment containing the GFP coding sequence and the 3¢ untransformed region (UTR) of unc-54 (from plasmid pPD95.75 kindly provided by A Fire) For gstk-1 (ZK1320.1), a 1.1 kb promoter fragment was amplified and fused with the GFP coding sequence and then coamplified with the gstk-1 genomic and 3¢ UTR Sequences were checked and the resulting gfp::gstk-1 and gstk-2::gfp fragments were microinjected [32] at 50 ngỈlL)1 into the syncytial gonad of young wild-type adult hermaphrodites, together with 200 ngỈlL)1 of the plasmid pRF4 containing the dominant marker rol-6(su1006) [33] For each construct, at least three independent lines were analysed for expression Immunofluorescence microscopy Routinely, fluorescence expression patterns and phenotypic analyses were carried out on a Zeiss axioskop plus equipped with Nomarski optics (Zeiss, Le Pecq, France) Confocal stacks of images every 0.3–0.5 lm were captured on an inverted Leica SP2 confocal microscope (Leica, Rueil-Malmaison, France) Z projections were analysed FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS 5037 Glutathione transferase kappa in C elegans E Petit et al using Image J software and then processed using Adobe PhotoshopÒ To stain mitochondria, animals were incubated for 10 with 10 lm of MitoTracker Red (Invitrogen Molecular Probes, Cergy Pontoise, France), and then moved to a fresh plate for h Worms were anaesthetized in either mgỈmL)1 levamisole or 10 lm azide RNAi experiments RNAi by feeding bacteria was performed using the N2 strain, as described previously [34,35], with the following modifications The bacterial HT115(DE3) E coli strains used for feeding experiments were obtained from J Ahringer (University of Cambridge, UK) (gstk-1, ref: WBRNAi00021881) and OpenBiosystems (Fisher Scientific-Open Biosystems, Illkirch, France) (gstk-2, ref : RCE1182) In brief, control and RNAi strain cultures were grown for h in LB medium containing 100 mgỈmL)1 ampicillin, and then spread onto NGM agar containing isopropyl thio-b-dgalactoside (1 mm) and carbenicillin (25 lgỈmL)1) For double-RNAi treatment, equal concentrations of both strains were mixed before seeding The next day, animals at the fourth larval stage were placed onto RNAi plates, grown for days and then harvested by rinsing with M9 buffer (0.1 m NaCl, 0.05 m potassium phosphate, pH 6.0) Adults were allowed to settle, and eggs were recovered from hermaphrodites by alkaline hypochlorite lysis (5 at room temperature in 0.5 m NaOH, 5% hypochlorite) [36] The eggs were rinsed with M9 buffer and the resulting L1 larvae were transferred the next day to fresh agar plates containing the different dsRNA conditions Lifespan assays First-generation progeny from RNAi and control conditions were picked at the fourth larval stage and transferred onto fresh RNAi plates The day of the shift was counted as day in the adult lifespan assay To prevent mixing test worms with their progeny during the reproduction period, adult nematodes were transferred daily to fresh plates Monitoring of lethality was performed every day and worms were considered to be dead when they failed to move, either spontaneously or in response to touch, and showed no pharyngeal pumping Worms that crawled off the plate were excluded (considered to have escaped) One hundred worms per condition were used in each lifespan experiment, conducted in triplicate Nematode survival was analysed by the Kaplan–Meier method using Graphpad Prism The same software was used to test the equality of survival with the log-rank (Wilcoxon) test Oxygen consumption assays Oxygen consumption rates were measured using a DW1 ⁄ AD Clark-type oxygen electrode (Hansatech, Norfolk, UK) 5038 Young adult worms that were maintained on NGM agar plates covered with the corresponding RNAi bacteria were washed twice and resuspended in 50 lL of M9, and then transferred into the chamber already containing 450 lL of M9 buffer, and respiration was measured at 20 °C for at least 10 All washes and measurements were performed in oxygenated M9 buffer Samples were carefully recovered from the chamber and the number of worms was counted For each condition, the mean rate was calculated from triplicate experiments Western blotting To prepare total extracts, worm pellets were resuspended in Laemmli sample buffer, vortexed three times for 15 s after the addition of broken glass beads, and then denatured for at 100 °C and separated by 10% SDS–PAGE Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany) and probed with the mouse anti-GFP IgG1k (Roche Diagnostics, Meylan, France) Immunoreactive proteins were revealed with a chemiluminescent detection system (SuperSignal Pico Chemiluminescent Substrate; Pierce Inc., Rockford, IL, USA) Lipid analyses Aliquots of C elegans were crushed in mL of methanol– mm EGTA (2 : 1, v ⁄ v) with an Ultra Turax; 100 lL of homogenate were evaporated and the pellet was dissolved in 0.25 mL of NaOH (0.1 m) overnight for protein measurement using the Bio-Rad assay For each analysis, lipids from the homogenate were extracted according to Bligh and Dyer [37] in chloroform–methanol–water (2.5 : 2.5 : 2.1, v ⁄ v ⁄ v) in the presence of the internal standards For total fatty acid analysis, lipids from a 200 lL homogenate were extracted and transmethylated with mL BF3 ⁄ CH3OH (SUPELCO 10% w ⁄ w) for h at 150 °C FAMEs were extracted with mL of hexane–1 mL of water The organic phase was evaporated to dryness and dissolved in 20 lL of ethyl acetate One microlitre of FAME was analysed by gas–liquid chromatography [38] on a 5890 Hewlett Packard system using a Famewax RESTEK fused silica capillary column (30 m · 0.32 mm inside diameter, 0.25 mm film thickness) The oven temperature was programmed from 110 to 220 °C at a rate of °CỈmin)1 and the carrier gas was hydrogen (0.5 bar) The injector and detector were maintained at 225 and 245 °C, respectively Finally, lg of glyceryl triheptadecanoate were used as internal standard For free fatty acid analysis, 400 lL of homogenate were extracted and dissolved in mL of hexane Free fatty acids were transmethylated in mL of BF3 ⁄ CH3OH (10% w ⁄ w) for at room temperature and free FAMEs were extracted with mL of hexane–1 mL of water The organic FEBS Journal 276 (2009) 5030–5040 ª 2009 The Authors Journal compilation ª 2009 FEBS E Petit et al phase was evaporated to dryness and dissolved in 10 lL of ethyl acetate Analysis was performed as above with lg of nonadecanoic acid as internal standard All chemicals were obtained from Sigma-Aldrich, Lyon, France Acknowledgements This work was supported in part by the Institut ´ ´ National de la Sante et de la Recherche Medicale, Centre National de la Recherche Scientifique and Association pour la Recherche sur le Cancer Elise Petit was founded by the Ligue National Contre le Cancer and Xavier Michelet by the Association pour la Recherche contre le Cancer We are grateful to Professor A Guillouzo and Drs E Culetto and B Fromenty for critical reading of the manuscript The Imaging and Cell Biology Facility of the IFR87 (FR-W2251) ‘La plante et son environnement’ is sup` ported by the Action de Soutien a la Technologie et la Recherche en Essonne, Conseil de l’Essonne References Harris JM, Meyer DJ, Coles B & Ketterer B (1991) A novel glutathione transferase (13-13) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes Biochem J 278, 137–141 Ladner JE, Parsons JF, Rife CL, Gilliland GL & 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BM883 827 .1) *Residues involved in glutathione binding site #Residues involved in dimer interface GSTK -1 and GSTK -2 are localized in peroxisomes and mitochondria, respectively The analysis of GST kappa. .. fatty acids 14 12 10 16 :0 18 :0 16 :1w7 FEBS Journal 27 6 (20 09) 5030–5040 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 18 :1w9 18 :1w7 18 :2w6 20 :5w3 17 Cyclo Fatty acid mono esters 5035 Glutathione. .. Journal 27 6 (20 09) 5030–5040 ª 20 09 The Authors Journal compilation ª 20 09 FEBS 50 31 Glutathione transferase kappa in C elegans A 69 gstk -1 (zk1 320 .1) B AA position rGSTK1 GSTK -1 GSTK -2 4 62 51 150 18 2