Báo cáo khoa học: Lpx1p is a peroxisomal lipase required for normal peroxisome morphology potx

Lpx1p is a peroxisomal lipase required for normal peroxisome morphology Sven Thoms 1, *, Mykhaylo O. Debelyy 1 , Katja Nau 1, †, Helmut E. Meyer 2 and Ralf Erdmann 1 1 Institut fu ¨ r Physiologische Chemie, Abteilung fu ¨ r Systembiochemie, Ruhr-Universita ¨ t Bochum, Germany 2 Medizinisches Proteomcenter, Ruhr-Universita ¨ t Bochum, Germany Peroxisomes are ubiquitous eukaryotic organelles that are involved in lipid and antioxidant metabolism. They are versatile and dynamic organelles engaged in the b-oxidation of long and very long chain fatty acids, in a-oxidation, bile acid and ether lipid synthe- sis, and in amino acid and purine metabolism [1]. Peroxisomes are a source of reactive oxygen species, and are involved in the synthesis of signalling mole- cules in plants. Remarkably, peroxisomes are the only site of fatty acid b-oxidation in plants and fungi. Human peroxisomal disorders can be categorized as either single-enzyme disorders or peroxisomal biogenetic defects [2]. Single-enzyme disorders, for example Refsum disease caused by a defect of phytanoyl CoA hydroxylase, or X-linked adrenoleu- kodystrophy caused by a defect in a peroxisomal ATP-transporter. Biogenetic defects are mostly caused by mutations in the peroxisomal biogenesis genes, the PEX genes, that code for peroxins [3]. Peroxi- somal disorders are associated with morphological Keywords lipase; peroxin; peroxisome; proteomics; PTS1 Correspondence R. Erdmann, Abteilung fu ¨ r Systembiochemie, Ruhr-Universita ¨ t Bochum, Universita ¨ tsstr. 150, 44780 Bochum, Germany Fax: +49 234 32 14266 Tel: +49 234 322 4943 E-mail: ralf.erdmann@rub.de Present address *Universita ¨ tsmedizin Go ¨ ttingen, Abteilung fu ¨ rPa ¨ diatrie und Neuropa ¨ diatrie, Georg- August-Universita ¨ t, Germany †Forschungszentrum Karlsruhe, Institut fu ¨ r Toxikologie und Genetik, Germany (Received 20 September 2007, revised 22 November 2007, accepted 30 November 2007) doi:10.1111/j.1742-4658.2007.06217.x Lpx1p (systematic name: Yor084wp) is a peroxisomal protein from Saccha- romyces cerevisiae with a peroxisomal targeting signal type 1 (PTS1) and a lipase motif. Using mass spectrometry, we have identified Lpx1p as present in peroxisomes, and show that Lpx1p import is dependent on the PTS1 receptor Pex5p. We provide evidence that Lpx1p is piggyback-transported into peroxisomes. We have expressed the Lpx1p protein in Escherichia coli, and show that the enzyme exerts acyl hydrolase and phospholipase A activ- ity in vitro. However, the protein is not required for wild-type-like steady- state function of peroxisomes, which might be indicative of a metabolic rather than a biogenetic role. Interestingly, peroxisomes in deletion mutants of LPX1 have an aberrant morphology characterized by intraperoxisomal vesicles or invaginations. Abbreviations BPC, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY-FL C 11 -PC); DGR, 1,2-O-dilauryl-rac-glycero-3-glutaric acid (6-methyl resorufin) ester; DPG, 1,2-dioleoyl-3-(pyren-1-yl)decanoyl-rac-glycerol; PNB, p-nitrobutyrate. 504 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS peroxisomal defects such as inclusions or invagin- ations [4,5]. Peroxisomal import of most matrix proteins depends on the PTS1 (peroxisomal targeting signal type 1) receptor Pex5p, which recognizes the PTS1 localized at the very C-terminus [6,7]. The three-amino-acid signal SKL (serine–lysine–leucine) was the first PTS1 to be discovered, and is in many cases sufficient for directing a protein to peroxisomes. Most PTS1 are tripeptides of the consensus [SAC][KRH][LM] located at the extreme C-terminus. A second matrix protein peroxisomal targeting sig- nal (PTS2) is present in considerably fewer peroxi- somal proteins. PTS2 is usually located within the first 20 amino acids of the protein, and has been defined as [RK][LVIQ]XX[LVIHQ][LSGAK]X[HQ][LAF] [8]. PTS2-bearing proteins are recognized by the cytosolic receptor Pex7p. Systems biology approaches led to the identification of Lpx1p as an oleic acid-inducible peroxisomal matrix protein of unknown function [9,10]. The gene sequence of LPX1 predicts a lipase motif of the GxSxG type that is typical for a⁄ b hydrolases [11,12]. Using mass spectrometry, we identify Lpx1p as present in peroxi- somes, and analyse its peroxisomal targeting. We show that it acts as a phospholipase A, and, by electron microscopy and morphometry, we provide the first evi- dence for an interesting peroxisomal phenotype of the Dlpx1 deletion mutant. Results Identification of Lpx1p in peroxisomes by mass spectrometry We identified Lpx1p (lipase 1 of peroxisomes; EC 3.1.1.x) in a follow-up study to an exhaustive pro- teomic characterization of peroxisomal proteins [13]. This involved purification of peroxisomes from oleic- acid induced Saccharomyces cerevisiae, and subsequent membrane extraction using low- and high-salt buffers. Low-salt-extractable proteins were solubilized in SDS buffer, and separated by RP-HPLC [14]. Proteins in individual HPLC fractions were further separated by SDS–PAGE, and protein bands were cut out and anal- ysed by mass spectrometry. Lpx1p (systematic name: Yor084wp) was extractable by low salt and identified together with the peroxisomal aspartate aminotransfer- ase Aat2p in HPLC fraction 7 at a molecular mass of approximately 45 kDa (Fig. 1A) [15]. The predicted molecular mass of Lpx1p is 44 kDa. It carries a peroxisomal targeting signal type 1, gluta- mine–lysine–leucine (QKL) (Fig. 1B,D). The amino acid sequence comprises the lipase motif GHSMG of the general GxSxG type [11,16] with the central serine being part of the catalytic triad. This lipase motif is indicative of a⁄ b hydrolase family members [12]. Hydrophobicity predictions [17] indicate a pronounced hydrophobic region in the central domain, consisting of amino acids 154–177 with the core region 164LLI- LIEPVVI173 (Fig. 1C). By homology searches with other prokaryotic and eukaryotic hydrolases (not shown) using profile hidden Markov models [18], we identified a conserved histi- dine that is probably part of the catalytic triad of the active site (Fig. 1B). The third member of the catalytic triad could not be identified by sequence-based searches. PTS1-dependent targeting of Lpx1p to peroxisomes The majority of the Lpx1p in a cell homogenate was pelleted at 25 000 g, consistent with an organellar localization of the protein (Fig. 2A). In this experi- ment, more of the peroxisomal soluble thiolase Fox3p (EC 2.3.1.x) than of Lpx1p appears to be present in the supernatant. This is probably due to partial peroxi- some rupture during preparation, and might indicate that Lpx1p, in contrast to Fox3p, is loosely associated with the peroxisomal membrane. The peroxisomal localization of Lpx1p had been demonstrated indirectly by immuno-colabelling of a heterozygous C-terminally Protein A-tagged version of Lpx1p in a diploid strain [10]. Peroxisomal locali- zation under these conditions would depend on the presence of copies of Lpx1p that are not blocked by a C-terminal tag, and by the interaction of Lpx1p with itself (piggyback import). We wished to analyse whether Lpx1p directly localized to peroxisomes, and cloned LPX1 for expression from a yeast shuttle plasmid using an N-terminal GFP tag. This fusion protein was localized to peroxisomes in a Dlpx1 dele- tion strain (Fig. 2B), indicating that Lpx1p by itself targets to peroxisomes. Peroxisomal localization of Lpx1p was abolished when Lpx1p was expressed with a C-terminal tag (Fig. 2C), indicating that the C-terminus has to be free for Pex5p-dependent import. Peroxisomal localization was abolished in the absence of Pex5p (Fig. 2C), and was not affected by the absence of Pex7p (Fig. 2C), indicating that its targeting to peroxisomes is dependent on the PTS1 pathway. We confirmed the peroxisomal localization of Lpx1p by subcellular fractionation. On a sucrose density gra- dient, GFP–Lpx1p co-migrated with Fox3p (alternative S. Thoms et al. Peroxisomal lipase Lpx1p FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 505 name: Pot1p), with Pex11p, and with the catalase (EC 1.11.1.6) activity peak in the same density fraction at about 1.225 gÆcm )3 (fraction 10) (Fig. 2D). The activ- ity of the mitochondrial marker fumarase (EC 4.2.1.2) together with the mitochondrial Mir1p showed a clearly separate peak at a density of 1.192 gÆcm )3 in fraction 14 (Fig. 2D). Lpx1p was identified from low-salt-extractable mem- branes (Fig. 1A), and the amount of Lpx1p that is not membrane-associated or found in the non-peroxisomal low-density fractions (Fig. 2D; fractions 19–29) is low compared to Fox3p. Although the QKL C-terminus of Lpx1p does not match the PTS1 consensus [SAC][KRH][LM], a QKL terminus is able to target a test substrate to peroxi- somes [19]. Lpx1p is one of four S. cerevisiae proteins that end in QKL (Fig. 1D), and is probably the only one that is localized to peroxisomes. Self-interaction of Lpx1p C-terminally tagged Lpx1p localizes only to peroxi- somes when endogenous copies of the protein are pres- ent [10]. This suggests piggyback import of Lpx1p, which, in turn, would rely on self-interaction of Lpx1p. We tested this hypothesis by two-hybrid analysis of LPX1. Neither the fusion of Lpx1p with the GAL4 binding domain nor its fusion with the activation domain were auto-activating (Fig. 3A). The strains expressing both fusions exhibit a strong two-hybrid interaction signal, exceeding that of the control PEX11 with PEX19 (Fig. 3A). Because complex formation Fig. 1. Identification of Lpx1p from Saccharomyces cerevisiae peroxisomes by proteomics. (A) Isolation of putative peroxisomal proteins by preparative chromatographic separation. Low salt- extractable peroxisomal proteins were solubilized by SDS and separated by reverse phase HPLC. Polypeptides of selected frac- tions were separated by SDS–PAGE and visualized by Coomassie blue staining. Only the first 13 lanes of the HPLC profile are shown [15]. The band marked by an asterisk contains the peroxisomal proteins Lpx1p (predicted molecular mass 44 kDa) and Aat2p (pre- dicted molecular mass 44 kDa) in HPLC fraction 7 at a molecular mass of approximately 45 kDa. (B) Alignment of the LPX1 gene with a Mycoplasma genitale (Mg) gene encoding a putative ester- ase ⁄ lipase (AAC71532) and with the putative triacylglycerol lipase AAB96044 from Mycoplasma pneumoniae (Mp). Identical amino acids are indicated by an asterisk and similar amino acids are indi- cated by a colon and full point, depending on degree of similarity. The conserved GxSxG lipase motif is shaded in grey. The lipase motif contains the putative active-site serine. The arrowhead indicates the probable active-site histidine, as determined from alignments using eukaryotic esterase lipase family members (not shown). The third member of the catalytic triad could not be identi- fied by sequence-based analysis. (C) Hydropathy plot of Lpx1p. A Kyte–Doolittle plot was calculated with window size of 11. Values > 1.8 may be regarded as highly hydrophobic regions. (D) Termini of all four QKL proteins from S. cerevisiae. Only Lpx1p is predicted to target to peroxisomes. Positions relative to the (putative) PTS1 are indicated. Grey boxes, lysine in position -1 and valine in position -5 are probably required to target Lpx1p to peroxisomes. Peroxisomal lipase Lpx1p S. Thoms et al. 506 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS might play a significant role in peroxisomal (piggyback) protein import [20], we determined the size of the Lpx1p complex by gel filtration of cell lysates of oleate-induced cells on a Superdex 200 column. We found that the majority of Lpx1p is not present in high-molecular- mass complexes, but elutes at molecular masses cor- responding to monomers, dimers and trimers (Fig 3B). The two-hybrid interaction probably reflects the com- plex formation. However, our identification of low- molecular-mass complexes of Lpx1 does not exclude the possibility that higher-molecular-mass complexes are transiently formed during topogenesis of the protein. Lpx1p is not required for peroxisome biogenesis Having shown that Lpx1p is targeted to peroxisomes by the soluble PTS1 receptor, we wished to determine whether Lpx1p is required for the biogenesis of peroxi- somes. We first tested the Dlpx1 knockout for growth on oleate. However, Lpx1p is dispensable for growth on oleate as the only carbon source (Fig. 4A). To determine the influence of Lpx1p on peroxisome bio- genesis in more detail, post-nuclear supernatants were prepared from wild-type and Dlpx1 strains. The post- nuclear supernatants were analysed by Optiprep gradient analysis and subsequent tests of gradient fractions for peroxisomal catalase and mitochondrial cytochrome c oxidase (EC 1.9.3.l; Fig. 4B). None of these marker pro- teins indicated a significant change in the abundance or density of peroxisomes or mitochondria, suggesting that peroxisomal and mitochondrial biogenesis remain functional after deletion of the LPX1 gene. Lipase activity of Lpx1p Characteristic GxSxG motifs and similarities with a ⁄ b hydrolases in the predicted protein sequence sug- gest that Lpx1p is an esterase, possibly a lipase [11,12,16]. To directly investigate Lpx1p, we expressed the full-length protein as a fusion protein with a C-ter- Fig. 2. Localization and PTS1-dependent targeting of Lpx1p to peroxisomes. (A) Immunological detection of GFP–Lpx1p in a sedimentation experiment. A cell-free homogenate (T) was separated into supernatant (S) and an organelle-containing pellet fraction (P) by centrifugation at 25 000 g (30 min). Amounts corresponding to equal T content of each fraction were analysed by SDS–PAGE and western blotting with antibodies against GFP and the peroxisomal marker protein oxoacyl CoA thiolase, Fox3p (alternative name: Pot1p). (B) Lpx1p is localized to peroxisomes. Coexpression of PTS2-dsRed and GFP–Lpx1p in yeast cells. Cells were grown on ethanol to induce the expression of PTS2- dsRed. (C) Import of Lpx1p into peroxisomes is dependent on Pex5p and independent of Pex7p. Lpx1p was expressed as either a C-terminal fusion (top images) or N-terminal fusion (bottom images) with GFP. In the Dpex5 deletion mutant, Lpx1p cannot be imported into peroxi- somes, irrespective of the position of the tag (right). Deletion of PEX7 does not influence Lpx1p targeting if the PTS1 is not blocked by GFP (top left). GFP fusion proteins that are not targeted to peroxisomes mislocalize to the cytosol. Bar = 2 lm. (D) Sucrose density gradient anal- ysis of GFP–LPX1-transformed yeast. A cell-free organelle sediment from oleate induced cells was analysed on a density gradient with sucrose concentrations form 32 to 54% w ⁄ v. Individual fractions were analysed for catalase activity (peroxisomal marker) and fumarase activity (mitochondrial marker). In addition, the presence of GFP–Lpx1p, Fox3p, Pex11p (peroxisomal membrane protein) and Mir1p (mito- chondrial phosphate carrier) was tested by western blotting and immunodetection. S. Thoms et al. Peroxisomal lipase Lpx1p FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 507 minal hexahistidine tag in Escherichia coli and purified the protein using immobilized metal-ion affinity chro- matography (Fig. 5A). The protein was further puri- fied by gel filtration on a Superdex 200 column (Fig. 5B). Gel filtration indicated the propensity of Lpx1p to oligomerize in vitro, albeit to a much lower extent than in yeast whole-cell lysates (compare Figs 3B and 5B). Purified protein was used for the generation of poly- clonal antibodies in rabbit. Antisera recognized a pro- tein of about 43 kDa, indicating that the antiserum is specific for Lpx1p. We used these antibodies to con- firm that the endogenous yeast Lpx1p is induced by oleic acid (Fig. 5A). To analyse the enzyme activity of Lpx1p, we assayed the E. coli-expressed protein for esterase activity, using p-nitrophenyl butyrate (PNB) as the test substrate. PNB can be hydrolysed by esterases, yielding free p-nitro- phenol, which can be determined photometrically at 410 nm. Lpx1p hydrolysed the test substrate with a K M of 6.3 lm and V max of 0.17 lmolÆs )1 (Table 1). Lpx1p is strongly induced by oleic acid, regulated by stress-associated transcription factors [21], and aligns with human epoxide hydrolases (EC 1.14.99.x; not shown). We found that Lpx1 hydrolysed the epoxide hydrolase substrate [22] 4-nitrophenyl-trans-2,3-epoxy- 3-phenylpropyl carbonate (NEPC) (data not shown), but we consider that this activity is non-specific, because it could not be blocked by the specific epoxide hydrolase inhibitor N,N’-dicyclohexylurea (DCU) (data not shown). To test for lipase activity, we used 1,2-dioleoyl-3- (pyren-1-yl)decanoyl-rac-glycerol (DPG) as a substrate. DPG contains the eximer-forming pyrene decanoic acid as one of the acyl residues. Upon cleavage, the free pyrene decanoic acid shows reduced eximer fluorescence. Lpx1p exerts lipase activity towards DPG of 5.6 pmolÆh )1 Ælg )1 (Table 1). For comparison, we measured the lipase activity of commercial yeast Candida rugosa lipase towards DPG and found an Fig. 3. Lpx1p interacts with itself. (A) Two-hybrid assay. Full-length Lpx1p was fused to the GAL4 binding or activation domain and co- expressed in a yeast strain with Escherichia coli b-galactosidase under the control of a GAL4-inducible promotor. b-galactosidase activity was measured in lysates of doubly transformed strains. No signal was obtained when LPX1 was combined with empty vectors. Positive control: interaction of Pex19p with Pex11p. (B) Size-exclusion chromatography of a wild-type cell lysate of oleate-induced cells. The lysate was fractionated by gel filtration on a Superdex 200 column and tested by immunoblotting with anti-Lpx1p antiserum. The molecular masses indicated were interpolated from a calibration curve and correspond well with monomeric, dimeric and trimeric forms of Lpx1p. The relative distribution of the three forms was quantified using NIH Image (National Institutes of Health, Bethesda, MD, USA). The elution vol- ume is indicated in millilitre. Fig. 4. Absence of pex phenotype in a Dlpx1 deletion. (A) Growth on plates with oleate as the only carbon source. Wild-type, Dlpx1 or Dpex1 control stains were spotted in equal cell numbers in series of 10-fold dilutions on oleate or ethanol plates. Absence of growth and oleic acid consumption (halo formation) indicates a peroxisomal defect. Control: growth assay on ethanol. (B) Optiprep density gradi- ent centrifugation analysis of postnuclear supernatants prepared from oleate-induced wild-type and Dlpx1 strains. All fractions were analysed using catalase (peroxisome) and cytochrome c oxidase (mitochondria) enzyme assays. The peroxisomal and mitochondrial densities were not measurably altered by LPX1 deletion. Peroxisomal lipase Lpx1p S. Thoms et al. 508 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS activity of 2.0 pmolÆh )1 Ælg )1 under the same assay con- ditions (Table 1). We sought to confirm lipase activity by testing Lpx1p in a clinical assay for pancreatic lipase. The assay uses the substrate 1,2-O-dilauryl-rac-glycero- 3-glutaric acid (6-methyl resorufin) ester (DGR) in a desoxycholate-containing buffer. Lpx1p did not hydro- lyse this substrate under the assay conditions (Table 1). Next we tested for phospholipase C activity in a coupled enzyme assay with phosphatidylcholine as the substrate. In this assay, phospholipase C converts phosphatidylcholine to phosphocholine and diacylglyc- erol. Alkaline phosphatase hydrolyses phosphocholine to form choline, which is then oxidized by choline oxidase to betaine and hydrogen peroxide. The latter, in the presence of horseradish peroxidase, reacts with 10-acetyl-3,7-dihydrophenoxazine to form fluorescent resorufin. This assay, as well as a similar assay for phospholipase D, gave negative results for Lpx1p (Table 1). Finally, we tested phospholipase A (EC 3.1.1.4) activity using the substrate 1,2-bis-(4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-sindacene-3-undecanoyl)- sn-glycero-3-phosphocholine (bis-BODIPY-FL C 11 -PC, BPC). BPC is a glycerophosphocholine with BODIPY dye-labeled sn-1 and sn-2 C 11 acyl chains. Cleavage reduces dye quenching and leads to a fluorecence increase at 530 nm upon excitation at 488 nm. Lpx1p exerts phospholipase A activity of 7.9 pmolÆh )1 Ælg )1 . As a control enzyme, we used commercial porcine pancreas lipase, which hydrolysed 195 pmolÆh )1 Ælg )1 . In summary, Lpx1p shows acyl esterase, lipase and phospholipase A activity towards PNB, DPG and BPC, respectively. Altered peroxisome morphology in deletion mutants of LPX1 Lastly, we analysed electron microscopic (EM) images of knockouts of LPX1. To our surprise, a large number of Dlpx1 peroxisomes showed an abnormal morphology. The peroxisomes appear vesiculated Fig. 5. Protein expression, antibody genera- tion and oleate induction of Lpx1p. Expres- sion of Lpx1p in Escherichia coli. (A) Lpx1p was expressed as a fusion protein with a C-terminal hexahistidine tag and purified by His-trap chromatography. The purified Lpx1p (lane 1) was used to generate polyclonal antibodies in rabbit that recognize the puri- fied recombinant protein (lane 4). Endoge- nous Lpx1p in whole yeast lysates is recognized only after induction of peroxi- somes and Lpx1p by oleate (lane 2 versus lane 3). Molecular masses are shown in kDa. (B) Second purification step: gel filtra- tion on Superdex 200 column. The elution profile indicates that most of the protein behaves as a monomer, but a small propor- tion forms dimers and trimers. Table 1. Esterase, lipase, and phospholipase activity of Lpx1p. Esterase activity was measured using PNB (p-nitrobutyrate) as a substrate. K M and V max values were calculated using Michaelis– Menten approximations. Lipase activity was determined using DPG as a substrate. Activity was measured from two independent pro- tein preparations in triplicate. Candida rugosa lipase (CRL) was used as a positive control for lipase measurement. (Pancreas) lipase activity assays used DGR in a coupled enzyme assay as a sub- strate. Phospholipase C and D (PLC and PLD) activities were mea- sured in coupled enzyme assays using phosphatidylcholine (PC). Phospholipase A measurements used BPC (bis-BODIPY-FL C 11 -PC) as a test substrate. Porcine pancreas lipase (PPL) was used as a control. Enzyme Substrate Activity Activity parameters (pmolÆh )1 Ælg )1 ) Lpx1p PNB Acyl esterase K M 6.3 lM; V max 0.17 lmolÆs )1 Lpx1p DPG (Triacylglycerol) lipase 5.6 ± 1.5 CRL DPG (Triacylglycerol) lipase 2.0 ± 0.1 Lpx1p DGR (Pancreas) lipase Below detection limit Lpx1p PC PLC Below detection limit Lpx1p PC PLD Below detection limit Lpx1p BPC PLA 7.9 PPL BPC PLA 195 S. Thoms et al. Peroxisomal lipase Lpx1p FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 509 (Fig. 6B), and either contain intraperoxisomal vesicles or their membrane is grossly invaginated. On average, one vesiculated peroxisome is visible in every fifth mutant cell (Fig. 6E). When the average number of altered peroxisomes is counted, we find that every third peroxisome shows this vesiculation phenotype (Fig. 6D). This is a high percentage, considering the fact that the peroxisomes were viewed in thin micro- tome sections. In three dimensions, every single peroxi- some might contain a vesicular membrane or indentation that escapes notice in two-thirds of the ‘two-dimensional’ sections. The average number of peroxisomes per cell is insignificantly increased in Dlpx1 (2.95 versus 2.76, Fig. 6C). Wild-type cells did not contain any vesicu- lated peroxisome (Fig. 6A,D,E). The drastic phenotype of Dlpx1 is reminiscent of the peroxisomal morphology found in peroxisomal disorders. Discussion Lpx1p is a peroxisomal protein with an unusual PTS1 LPX1 is one of the most strongly induced genes fol- lowing a shift from glucose to oleate, as determined by serial analysis of gene expression (SAGE) experiments [9]. The oleate-induced increase in mRNA abundance is abolished in the Dpip2 Doaf1 double deletion strain, indicating that its induction is dependent on the tran- scription factor pair Pip2p and Oaf1p [9]. The Lpx1p protein itself is induced by oleic acid as determined using a Protein A tag [10] or by use of an antibody raised against Lpx1p (see Results). Lpx1p does not conform to the general PTS1 con- sensus. The other three QKL proteins in S. cerevisiae are probably not peroxisomal (Fig. 1D): Efb1p (systematic name: Yal003wp) is the elongation factor EF-1b [23], Rpt4p (Yor259cp) is a mostly nuclear 19S proteasome cap AAA protein [24], and Tea1p (Yor337wp) is a nuclear Ty1 enhancer activator [25]. However, QKL is sufficient to sponsor Pex5p binding [19]. Why are these QKL proteins not imported into peroxisomes? This is probably due to the upstream sequences. Lpx1p has a lysine at position -1 (relative to the PTS1 tripeptide) and a hydrophobic amino acid at position -5 (Fig. 1D). These features promote Pex5p binding and are not found in the other three QKL proteins (Fig. 1D) [19]. Our views were confirmed by applying a PTS1 prediction algorithm (http:// mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp) [26], which predicted peroxisomal localization for Lpx1p only of the four proteins listed in Fig. 1D. Fig. 6. Peroxisome morphology phenotype of the Dlpx1 deletion. Absence of LPX1 leads to drastic peroxisomal vesiculation or invagi- nation. Electron microscopic images of cells from (A) wild-type and (B) Dlpx1. All cells were grown on medium with 0.1% oleic acid. Per- oxisomes are marked by arrowheads. Bar = 2 lm. (C) Comparison of per cell peroxisome numbers in wild-type and Dlpx1 strains. (D) Aver- age number of vesicles per peroxisome (wild-type, n = 94; Dlpx1, n = 142). In Dlpx1, about every third peroxisome contains a vesicle. (E) Percentage of cells with vesicle-containing peroxisomes. Roughly one in five Dlpx1 cells carries peroxisomes with a vesicle or invagi- nations (wild-type, n = 34; Dlpx1, n = 48). px, peroxisome(s). Peroxisomal lipase Lpx1p S. Thoms et al. 510 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS Lipase activity and cellular function of Lpx1p Lpx1p could be involved in various processes: (a) detoxification and stress response, (b) lipid mobiliza- tion, or (c) peroxisome biogenesis. As Lpx1p expres- sion may be regulated by Yrm1p and Yrr1p [21], a transcription factor pair that mediates pleiotropic drug resistance effects, we speculate that Lpx1p is required for a multidrug resistance response that did not show a phenotype in our experiments. We could, however, exclude epoxide hydrolase activity for Lpx1p, because hydrolysis of the epoxide hydrolase test substrate was not affected by a specific epoxide hydrolase inhibitor. We investigated the dimerization of Lpx1p in the context of piggyback protein import into peroxisomes. Self-interaction (dimerization) is frequently found in regulation of the enzymatic activity of other lipases such as C. rugosa lipase or human lipoprotein lipase [27,28]. The putative active-site serine of Lpx1p is located next to the region of highest hydropathy, sug- gesting that Lpx1p is a membrane-active lipase that contributes to metabolism or the membrane shaping of peroxisomes. Peroxisomes are sites of lipid metabolism. It is thus not surprising to find a lipase associated with peroxi- somes. Our experiments show that Lpx1p has triacyl- glycerol lipase activity; however, activities towards the artificial test substrates DPG and DGR were low. Our evidence for phospholipase A activity of the enzyme, together with the EM phenotype, suggest that Lpx1 has a more specialized role in modifying membrane phospholipids. Recently, a mammalian group VIB calcium-indepen- dent phospholipase A2 (iPLA 2 c) was identified that possesses a PTS1 SKL and a mitochondrial targeting signal [29,30]. The enzyme is localized in peroxisomes and mitochondria, and is involved, among others, in arachinoic acid and cardiolipin metabolism [31,32]. Knockout mice of iPLA 2 c show mitochondrial ⁄ cardio- logical phenotypes [33]. It will be exciting to determine whether human iPLA 2 c and yeast Lpx1p are function- ally related. We have provided evidence that peroxisomes are still functional in the absence of LPX1. This suggests a non-essential metabolic role for Lpx1p in peroxisome function. The morphological defect found in electron microscopic images of a deletion of Lpx1p (peroxisomes containing inclusions or invaginations) is symptomatic of a yeast peroxisomal mutant, and is reminiscent of the phenotypes found in human peroxi- somal disorders [4,5]. Out data suggest that Lpx1p is required to determine the shape of peroxisomes. Experimental procedures Strains and expression cloning The S. cerevisiae strains BY4742, BY4742 Dyor084w, BY4742 Dpex5, BY4742 Dpex7 and BY4742 Dpex1 were obtained from EUROSCARF (Frankfurt, Germany). S. ce- revisiae strain BJ1991 (Mata leu2 trp1 ura3-251 prb1-1122 pep4-3) has been described previously [34]. Genomic S. cerevisiae DNA was used as a PCR template for PCR. For construction of pUG35-LPX1 (LPX1–GFP), PCR-amplified YOR084w (primers 5¢-GCTCTAGAATG GAACAGAACAGGTTCAAG-3¢ and 5¢-CGGAATTCCA GTTTTTGTTTAGTCGTTTTAAC-3¢) was subcloned into EcoRV-digested pBluescript SK + (Stratagene, La Jolla, CA, USA), and then introduced into the XbaI and EcoRI sites of pUG35 (HJ Hegemann, Du ¨ sseldorf, Germany). For construction of pUG36-LPX1 (GFP–LPX1), PCR-amplified YOR084w (primers 5¢-GAGGATCCATGGAACAGAACA GGTTCAAG-3¢ and 5¢-CGGAATTCTTACAGTTTTTGT TTAGTCGTTTTAAC-3¢) was subcloned into EcoRV- digested pBluescript SK + , and then cloned into the BamHI and EcoRI sites of pUG36 (HJ Hegemann). pET21d-LPX1 was constructed by introducing PCR- amplified YOR084w (primers 5¢-GAATCCATGGAACAG AACAGGTTCAA-3¢ and 5¢-CGGTACCGCGGCCGCCA GTTTTTGTTTAGTCGTTTT-3¢) into the NcoI and NotI sites of pET21d (EMD Chemicals, Darmstadt, Germany). For construction of pPC86-LPX1 and pPC97-LPX1, YOR084w was amplified using primers 5¢-CCCGGGAAT GGAACAGAACAGGTTCAAG-3¢ and 5¢-AGATCTTTA CAGTTTTTGTTTAGTCGTTTT-3¢, and introduced into pGEM-T (Promega, Mannheim, Germany). The ORF was excised using XmaI and BglII, and introduced into pPC86 and pPC97 [35]. All constructs were confirmed by DNA seq- uencing. pPTS2-DsRed has been described previously [36]. Image acquisition Samples were fixed with 0.5% w ⁄ v agarose on microscopic slides. Fluorescence microscopic images were recorded on an Axioplan2 microscope (Zeiss, Ko ¨ ln, Germany) equipped with an aPlan-FLUAR 100 x ⁄ 1.45 oil objective and an AxioCam MRm camera (Zeiss) at room temperature. If necessary, contrast was linearly adjusted using the image acquisition software Axiovision 4.2 (Zeiss). Protein purification and antibody generation Lpx1p was expressed from pET21d-LPX1 in BL21(DE3) E. coli. Cells were harvested by centrifugation (SLA3000, 4000 g, 15 mins), and resuspended in buffer P (1.7 mm potassium dihydrogen phosphate, 5.2 mm disodium hydro- gen phosphate, pH 7.5, 150 mm sodium chloride) containing S. Thoms et al. Peroxisomal lipase Lpx1p FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS 511 a protease inhibitor mix (8 lm antipain-dihydrochloride, 0.3 lm aprotinin, 1 lm bestatin, 10 l m chymostatin, 5 lm leupeptin, 1.5 lm pepstatin, 1 mm benzamidin, and 1 mm phenylmethane sulfonylfluoride) and 50 l g Æ mL )1 lysozyme, 22.5 lgÆmL )1 DNAse I and 40 mm imidazole. Cells were sonicated 20 times for 20 s each using a 250D Branson digital sonifier (Danbury, CT, USA) with an amplitude setting of 25%. After removal of cell debris (SS34, 27 000 g, 45 min) the supernatant was clarified by 0.22 lm filtration (Sarstedt, Nu ¨ mbrecht, Germany) and loaded on Ni-Sepharose columns (GE Healthcare, Munich, Germany) equilibrated with buffer W (buffer P containing 300 mm sodium chloride, 1 mm dithiothreitol, 40 mm imidazole). The column was washed in buffer W until no further protein was eluted. Recombinant Lpx1p was eluted by a continuous 40–500 mm imidazole gradient based on buffer W. Peak fractions (identified by SDS–PAGE) were pooled and concentrated using VivaSpin concentrators (30 kDa cutoff, Sartorius, Go ¨ ttingen, Germany). Lpx1p was further purified by gel-filtration chromatography. Protein was stored at 0 °C. For the production of poly- clonal antibodies, gel bands corresponding to 150 lg protein were excised and used for rabbit immunization (Eurogentec, Seraing, Belgium). Size-exclusion chromatography For analysis of endogenous Lpx1p by gel filtration, 5 mL of a glass bead lysate of oleate-induced BY4742 wild-type cells in buffer A (buffer P, pH 7.3, 300 mm sodium chloride) with a protease inhibitor mix were injected into a HiLoad 16 ⁄ 60 Superdex 200 prepgrade column (GE Healthcare) and eluted using buffer A at a flow rate of 1 mL )1 Æmin and a fraction size of 2 mL. Fractions were analysed by SDS–PAGE and Western blotting. A 500 lL aliquot of the concentrated Ni-Sepharose eluate of Lpx1p from E. coli expression was purified in the same buffer under the same conditions. For estimation of Lpx1p complex sizes, molecular masses were interpolated from a calibration curve generated using ovalbumin (45 kDa), carboanhydrase (29 kDa), trypsin inhibitor (20.1 kDa), lactalbumin (14.2 kDa) and aprotinin (6.5 kDa) as molecular mass standards. Enzyme assays Esterase activity was determined using 0.5 mm p-nitrophe- nyl butyrate (Sigma-Aldrich, Seelze, Germany) in NaCl ⁄ P i (pH 7.4) in a total volume of 200 lLat37°C. The amount of free p-nitrophenol was determined at 410 nm in 96-well plates. Michaelis–Menten kinetics were analysed using GraphPad Prism4 (Graph Pad Software, San Diego, CA, USA). Lipase activity was determined using 0.5 mm DPG (Mar- ker Gene Technologies, Eugene, OR, USA) in 0.1 m gly- cine, 19 mm sodium deoxicholate, pH 9.5, in a total volume of 200 lLat37°C. Hydrolysis of DPG was followed in 96-well plates at 460 nm with 360 nm excitation using a Sirius HT fluorescence plate reader (MWG Biotech, Ebersberg, Germany). Lipase activity towards DPG was measured in assay setups containing 2–10 lg Lpx1p (from two independent protein preparations), with C. rugosa triacylglycerol lipase (Lipase AT30 Amano, 1440 UÆmg )1 , Sigma-Aldrich) as a control. Phospholipase A activity was measured using bis- BODIPY FL C 11 -PC (Molecular Probes ⁄ Invitrogen, Eugene, OR, USA) as the substrate. The assay setup con- tained 70 lg Lpx1p in 50 lL assay buffer (50 mm Tris, 100 mm sodium chloride, 1 mm calcium carbonate, pH 8.9) together with 50 lL substrate-loaded liposomes. Liposomes were prepared by injecting 90 lL of an ethanolic mixture of 3.3 mm dioleyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3.3 mm dioleyl phosphatidylglycerol (Avanti Polar Lipids) and 0.33 mm bis-BODIPY FL C 11 - PC into 5 ml assay buffer. Substrate turnover was mea- sured at 528 nm emission after 488 nm excitation. Activity was calculated from the initial velocity. Porcine pancreas phospholipase A 2 (Fluka ⁄ Sigma-Aldrich, Buchs, Swizer- land) was used as a control. Density gradient centrifugation Gradient centrifugation was carried out essentially as described previously [37]. Briefly, oleate-induced yeast cells were converted to spheroblasts using 25 UÆg )1 Zymoly- ase 100T (MP Biomedicals, Illkirch, France). Spheroblasts were gently ruptured by Potter–Elvehjem homogenization, and centrifuged at low speed (3 · 10 min at 600 g)to generate postnuclear supernatants. These supernatants, con- taining 5 mg protein, were loaded on a 32–54% sucrose gradient (Fig. 2D) or an Optiprep gradient (Fig. 4B) and centrifuged for 3 h at 19 000 g (Sorvall SV288, 19 000 rpm, 4 °C). The gradient was fractionated into about 29 frac- tions of 1.2 mL. 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Meyer HE (1998) Identification of phosphorylated proteins from thrombin-activated human platelets isolated by two-dimensional gel electrophoresis by electrospray ionization–tandem mass spectrometry (ESI-MS ⁄ MS) and liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) Electrophoresis 19, 1015–1023 FEBS Journal 275 (2008) 504–514 ª 2008 The Authors Journal compilation ª 2008 FEBS . November 2007) doi:10.1111/j.1742-4658.2007.06217.x Lpx1p (systematic name: Yor084wp) is a peroxisomal protein from Saccha- romyces cerevisiae with a peroxisomal targeting signal type 1 (PTS1) and a lipase. control for lipase measurement. (Pancreas) lipase activity assays used DGR in a coupled enzyme assay as a sub- strate. Phospholipase C and D (PLC and PLD) activities