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Biochem. J. (2013) 449, 195–207 (Printed in Great Britain) 195 doi:10.1042/BJ20120911 Pex5p stabilizes Pex14p: a study using a newly isolated pex5 CHO cell mutant, ZPEG101 Ryuichi NATSUYAMA*, Kanji OKUMOTO*† and Yukio FUJIKI*†1 *Graduate School of Systems Life Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, and †Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Pex5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5-deficient CHO (Chinesehamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known pex5 CHO mutants in its Pex5p expression. An undetectable level of Pex5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type Pex5pL, a longer form of Pex5p, suggesting a role for Pex5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various Pex5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in Pex5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that Pex5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no Pex5p would be a useful tool for investigating Pex5p function and delineating the mechanisms underlying peroxisomal matrix protein import. INTRODUCTION understanding of multiple functions of mammalian Pex5p. In mammals, two types of Pex5p isoforms have been identified: a shorter one (Pex5pS) and a longer one (Pex5pL) with a 37amino acid insertion at the N-terminal region [9,10]. By using a Pex5 CHO mutant, ZP105, defective in both PTS1 and PTS2 import due to the mutation in the PEX5 gene yielding unstable Pex5p [10,13], both isoforms of Pex5p are shown to function in protein import via PTS1 by transporting its cargo PTS1 proteins into peroxisomes by binding to its initial target Pex14p [13]. Furthermore, Pex5pL is indispensable for PTS2 protein import by specific interaction with Pex7p via the additional insertion and the proximal region to translocate the Pex7p–PTS2 protein complex to the peroxisome [13–15]. The essential role of Pex5pL in PTS2 protein import is evidently demonstrated with a Pex5 CHO cell mutant, ZPG231, where the impaired PTS2 protein import is restored only by Pex5pL [14]. We isolated another distinct mutant, ZP139, where only PTS1 protein, but not PTS2 protein, import is abrogated owing to the impaired binding of Pex5pL to PTS1 proteins [10,13]. Pex5p releases PTS1 cargos into the peroxisome matrix in collaboration with peroxisomal translocation machinery comprising of Pex14p, Pex13p and three RING peroxins [2,4,16]. Pex5p is then exported into the cytosol by ternary complexes comprising of Pex1p and Pex6p and their anchoring protein Pex26p, thereby acting as a shuttling receptor between peroxisomes and the cytosol [17,18]. In the present study, we isolated a novel PEX5 CHO mutant, ZPEG101, with a phenotype showing complete deficiency of Pex5p and affecting Pex14p stability. We also address a novel role for Pex5p in Pex14p stability via the fifth pentapeptide motif. Peroxisomes are ubiquitous intracellular organelles found in organisms ranging from yeast to humans. Peroxisomes function in a wide variety of metabolic pathways, including β-oxidation of very long chain fatty acids and biosynthesis of plasmalogentype ether-glycerolipids [1]. The functional significance of human peroxisomes is highlighted by fatal human genetic diseases, named PBDs (peroxisome biogenesis disorders), such as Zellweger syndrome [2,3]. To elucidate peroxisome biogenesis and human PBDs, more than 15 different CGs (complementation groups) of peroxisome-deficient mutants have been isolated from CHO (Chinese-hamster ovary) cells [2]. Genetic complementation analysis using peroxisome-deficient mutants of CHO cells as well as yeast species led to identification of a number of PEX genes essential for peroxisome biogenesis [2–4]. The majority of peroxisomal matrix proteins harbour a cisacting PTS1 (peroxisome-targeting signal type 1) and a Cterminal tripeptide SKL motif [5,6], with a few possessing a cleavable N-terminal presequence PTS2 [7,8]. Pex5p and Pex7p have been further identified as cytosolic receptors for PTS1 and PTS2 respectively [4]. PEX5 and PEX7 are shown to be causal genes for the PBDs of CG2 and CG11 respectively [2,3]. In mammals Pex5p recognizes PTS1 with seven TPR (tetratricopeptide repeat) domains in the C-terminal region [9–11] and interacts with the PMPs (peroxisomal membrane proteins) Pex14p and Pex13p via several pentapeptide WXXXF/Y motifs in the N-terminal portion [11,12]. In our collection of peroxisome-deficient CHO mutant cells [2], several Pex5 CHO mutants representing distinct phenotypes have contributed to the Key words: Chinese-hamster ovary cell mutant (CHO cell mutant), matrix protein import, peroxisome biogenesis, Pex14p, peroxisome-targeting signal type 1 (PTS1), PTS1 receptor (Pex5p). Abbreviations used: AOx, acyl-CoA oxidase; CG, complementation group; CHO, Chinese-hamster ovary; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; HA, haemagglutinin; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; PBD, peroxisome biogenesis disorder; Pex5p, peroxisome-targeting signal type 1 receptor; PMP, peroxisomal membrane protein; PMP70, 70 kDa integral PMP; P9OH, 9-(1 -pyrene)nonanol; PTS, peroxisome-targeting signal; RT, reverse transcription; siRNA, small interfering RNA; TPR, tetratricopeptide repeat. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2013 Biochemical Society 196 R. Natsuyama, K. Okumoto and Y. Fujiki EXPERIMENTAL Mutation analysis Antibodies mRNA was purified from of CHO-K1 and ZPEG101 cells (8×106 cells each) using a QuickPrep micro mRNA Purification Kit (GE Healthcare) according to manufacturer’s instructions. To amplify the entire open reading frame of PEX5 RT (reverse transcription)– PCR was performed with 5 μg of mRNA, SuperscriptIII reverse transcriptase (Invitrogen) and a pair of ClPEX5-specific PCR primers, a sense f1 and an antisense r1 primer (Table 1), as described previously [26]. PEX5 cDNA was cloned into pcDNAZeo3.1 (Invitrogen) and the nucleotide sequence was determined with a BigDye terminator cycle sequencing kit and a 3100-AVANT sequencer (Applied Biosystems). The present study used rabbit antibodies against AOx (acyl-coA oxidase) [19], 3-ketoacyl-CoA thiolase (thiolase) [19], catalase [19], cytochrome P450 reductase (Santa Cruz Biotechnology), Pex13p [20], Pex14p [21], PMP70 (70 kDa integral PMP) [19] and MDH (malate dehydrogenase) [22], and affinity-purified IgG against Pex5p [13]. Mouse monoclonal antibodies against FLAG (Sigma), influenza virus HA (haemagglutinin; 16B12, Covance), and His6 (BioDynamics Laboratory) and goat antiserum against LDH (lactate dehydrogenase; Rockland) were also used. DNA construction of PEX5 variants Cell culture and DNA transfection Wild-type CHO-K1, TKaEG1 and peroxisome-deficient CHO mutants, including ZPEG101, ZP105, ZP139 [10] and ZP110 [21] were cultured in Ham’s F-12 medium (Invitrogen) and HeLa cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen), both supplemented with 10 % FBS (fetal bovine serum) under 5 % CO2 /95 % air. DNA transfection into CHO cells was done using LipofectamineTM (Invitrogen) as described previously [23]. To obtain a parent cell for mutant isolation, TKa cells, wild-type CHO-K1 cells transformed with rat PEX2 cDNA [24], were transfected with a plasmid encoding EGFP (enhanced green fluorescent protein)–PTS1 in a pUcD2Hyg vector [25] and selected in the presence of 200 μg/ml hygromycin B (Sigma). A stable transformant showing the peroxisomal localization of EGFP–PTS1, termed TKaEG1, was cloned by limiting dilution method as described previously [26]. A stable transformant of ZPEG101 expressing N-terminally His- and C-terminally HA-tagged Chinese hamster Pex5pL (His-ClPex5pL-HA) was likewise isolated by transfection of pcDNAZeo/His-ClPEX5L-HA [27] followed by selection with Zeocin (Invitrogen) as described previously [23]. To construct the PEX5L(1–243) variants in pcDNA3.1/Zeo, PEX5L(1-243)Mut123, PEX5L(1-243)Mut234 and PEX5L(1243)Mut1234 in pGEX6P-1 [12] were cleaved by Sse8387INotI and replaced into wild-type pcDNAZeo/His–ClPEX5L– HA [27]. ClPEX5L variants were constructed by a PCRbased method using three sets of forward and reverse primers, Mut5f and AxyIr, Mut56f and Mut7r, and Mut7f and AxyIr, by using pcDNAZeo/His–ClPEX5L–HA as a DNA template. These PCR products were likewise amplified by a second PCR, using Mut56f-Mut7r and Mut7f-AxyIr as a template. The respective PCR products were cleaved with SacI and AxyI and cloned into the wild-type ClPEX5L, PEX5LMut123 and PEX5LMut1234 [12] using a pcDNA3.1/Zeo vector by replacing their corresponding fragments. To construct the deletion variants of PEX5L-FLAG encoding Pex5pL(1-305) and Pex5pL(306-632), PCR was performed with two primer pairs, T7 and 306stop_r and 307f and 632r, using pcDNAZeo/His–ClPEX5L–FLAG [27] as a template. The BglII–XhoI fragments of the respective PCR products were cloned into pcDNAZeo by replacing the BamHI– XhoI fragment of pcDNAZeo/His–ClPEX5L-FLAG. Northern blot analysis Isolation of a peroxisome-deficient CHO mutant ZPEG101 Peroxisome-deficient mutants were isolated using TKaEG1 as a parent cell using the same procedure as for the other well-established parental cell, TKaEG2, a TKa cell stably expressing PTS2–EGFP [26]. TKaEG1 cells were mutagenized with 0.4 μg/ml of ICR-191 (Sigma) for 7 h and P9OH [9-(1 pyrene)nonanol]/UV-resistant colonies defective in peroxisome assembly were selected by directly observing the intracellular localization of EGFP–PTS1 using a Carl Zeiss Axioskop FL microscope as described previously [26]. mRNA of the wild-type CHO-K1 and ZPEG101 cells was separated by electrophoresis, transferred on to a zeta-probe membrane (GE Healthcare) and hybridized with a 32 P-labelled BamHI fragment of ClPEX14 cDNA in ExpressHyb hybridization solution (Clontech) as described previously [21]. The membrane was washed twice at room temperature (22 ◦ C) and three times at 65 ◦ C with 2× SSPE [20 mM sodium phosphate (pH 7.4), 0.3 M NaCl and 1 mM EDTA] and 1 % SDS. Radioactive bands were detected by a FLA-5000 Autoimaging analyser (Fuji Film). The membrane was repeatedly hybridized with 32 P-labelled actin cDNA as a control for the loading and integrity of RNA. siRNA (small interfering RNA) Morphological analysis Immunostaining of CHO cells was performed as described previously [23], using 4 % paraformaldehyde for cell fixation and 0.1 % Triton X-100 for permeabilization. To verify the peroxisomal localization of the Pex5p variants, cells were semipermeabilized with 25 μg/ml of digitonin, washed to remove cytosolic Pex5p and then subjected to immunostaining as described previously [27]. Antigen–antibody complexes were visualized with Alexa Fluor® 488-, 568- or 647-labelled goat antibodies against rabbit or mouse IgG (Invitrogen) and observed together with EGFP fluorescence using a Axioplan2 fluorescent microscope and a LSM510 confocal laser microscope (Carl Zeiss). c The Authors Journal compilation c 2013 Biochemical Society Knockdown of Pex5p in HeLa cells was performed using a Stealth siRNA duplex (Invitrogen) specific to human PEX5 (target sequence, 5 -GGCAGAGAATGAACAAGAACTATTA3 ). Stealth RNAi negative control was also used as a control. HeLa cells were transfected twice with a 48-h interval with siRNA duplex and LipofectamineTM 2000 as described previously [27]. Subcellular fractionation and immunoprecipitation Cells were homogenized in homogenizing buffer [0.25 M sucrose, 20 mM Hepes-KOH (pH 7.4), 25 μg/ml each of leupeptin and antipain, and 1 mM PMSF] with a Potter–Elvehjem Teflon homogenizer and fractionated as described previously [13]. A novel CHO mutant lacking Pex5p Table 1 197 Primers and the sequences used in the present study f and r indicate forward and reverse primers respectively. Underline shows recognition sites of restriction enzymes and nucleotides for amino-acid substitutions or characteristic codons. Name Sequence (5 →3 ) Notes f1 r1 Mut5f Mut56f GCGCAGATCTTGGTCACCATGGCAATGAGGGAGCTG GCGCGCGGCCGCAGGGAGTCACATCCAGAGCGAGAAC CGGGCCGAGCTCAGGCAGAACAGGCGGCAGCAGAGGCTATACAGCAGCAG GTCGGGCCGAGCTCAGGCAGAACAGGCGGCAGCAGAGGCTATACAGCAGC AGGGCACATCAGAGGCCGCGGTCGATCAGGCCACAAGGTCAG CACCCCGCGCTTTCTGACGCTGATGACCTC CATCAGCGTCAGAAAGCGCGGGGTGCGCC AGCCCTTCTTCGAAAGCC CCGCTCGAGTCAATAGTCAGAAAGCCAGGG GGAAGATCTCCGATGACCTCACATCTGC CCGCTCGAGCTGGGGCAGGCCAAACATAGC BglII site, initiation codon NotI site, termination codon SacI site, W244A and F248A mutations SacI site, W244A, F248A, W258A, and Y262A mutations W301A and Y305A mutations W301A and Y305A mutations Mut7f Mut7r AxyIr 306stop_r 307f 632r For immunoprecipitation of FLAG-tagged proteins, cells or subcellular fractions were lysed in buffer-L [20 mM HepesKOH (pH 7.4), 150 mM NaCl, 25 μg/ml each of leupeptin and antipain, 1 mM PMSF, 1 mM EDTA, and 1 mM dithiothreitol containing 0.5 % CHAPS]. A soluble fraction was subjected to immunoprecipitation with anti-FLAG IgG-conjugated agarose (Sigma) as described previously [23]. Proteins bound to the beads and total cell lysates were analysed by SDS/PAGE (9 % gel) and immunoblotting as described previously [23]. Pulse–chase experiments CHO-K1, ZPEG101 and ZP110 cells growing in 35-mm-diameter dishes were washed twice with PBS and incubated in cysteine- and methionine-free DMEM (Invitrogen) for 30 min, and then pulselabelled for 1 h with 10 mCi/ml [35 S]methionine plus [35 S]cysteine (American Radiolabeled Chemicals). To chase the 35 S-labelled proteins, cells were washed with PBS and further incubated with complete Ham’s F12 medium containing 10 % FBS. At selected intervals, the cells were lysed in buffer-L containing 0.5 % Nonidet P40 and 0.1 % SDS. Soluble fractions were subjected to immunoprecipitation using an anti-Pex14p antibody as described previously [19]. In subcellular fractionation of 35 S-labelled cells, cells pulse-labelled for 1 h as described above were harvested and incubated in homogenizing buffer containing 25 μg/ml digitonin for 5 min at room temperature as described previously [28]. After centrifugation at 100 000 g for 30 min at 4 ◦ C, cytosolic and organellar fractions were subjected to immunoprecipitation with anti-Pex14p antibody as described above. 35 S-labelled proteins were separated by SDS/PAGE (9 % gel) and detected with a FLA5000 Autoimaging analyser (Fuji Film). Integrity of Pex14p Organelle fractions prepared as described above were resuspended in homogenizing buffer and divided into several equal aliquots (100 μl). They are added with various concentration of NaCl and incubated on ice for 30 min. These organelle fractions were re-centrifuged at 100 000 g for 30 min at 4 ◦ C and the pellet fractions were subjected to immunoblotting as described above. Other methods Protein bands in the immunoblot analysis were quantified with ImageJ (http://rsbweb.nih.gov/ij/). Statistical significance was examined by Student’s t test and shown as *P < 0.05 and **P < 0.01. XhoI site BglII site XhoI site RESULTS Isolation of a novel peroxisome-deficient mutant ZPEG101 To investigate the molecular mechanism of peroxisome biogenesis, we attempted to isolate peroxisome biogenesisdefective CHO cell mutants by mutagenesis using ICR-191 [26] and P9OH/UV selection methods [29]. As a parental cell, we used TKaEG1, which are CHO-K1 cells stably expressing rat Pex2p and EGFP–PTS1. EGFP–PTS1 in TKaEG1 was discernible in numerous punctate structures (Figure 1A, a) colocalizing with the PMP Pex14p (Figure 1A, c and e), indicative of the peroxisomal location. About 1.0×107 of TKaEG1 cells were mutagenized. After the treatment with P9OH/UV, viable cell colonies were examined for the intracellular localization of EGFP–PTS1, as was also done for the TKaEG2 cells [26]. Finally, a peroxisomedeficient mutant was isolated and named ZPEG101. ZPEG101 cells showed a diffused pattern of EGFP–PTS1 in the cytosol (Figure 1A, b), indicating a defect in peroxisomal PTS1 import. Interestingly, the Pex14p-positive structures in ZPEG101 were significantly less in fluorescence intensity and moderately smaller in number (Figure 1A, d) as compared with those in TKaEG1 (Figure 1A, c). In contrast, PMP70-positive membrane remnants, which are typically larger in size and smaller in number in pex mutants, were clearly discernible in ZPEG101 (Figure 1A, h) showing a fluorescence intensity similar to normal peroxisomes in TKaEG1 (Figure 1A, g). Pex14p was co-localized with the PMP70-positive structures in both TKaEG1 and ZPEG101 (Figure 1A, g and i, and h and j respectively) with the Pex14p level being distinctly lowered in ZPEG101 (Figure 1A, j). These results strongly suggested that ZPEG101 was a peroxisomal matrix protein import-defective mutant with a severely lowered level of Pex14p. To determine a CG of ZPEG101, 14 different PEX cDNAs were separately transfected into ZPEG101. EGFP–PTS1 import in ZPEG101 was restored only by expression of PEX5L (Figure 1B, b) and not other genes, including PEX1 (Figure 1B, a), PEX14 (Figure 1B, c), PEX2, PEX3, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX16, PEX19 and PEX26 (results not shown), strongly suggesting that ZPEG101 was a pex5 mutant. In immunostaining analysis, PTS1 protein, AOx, PTS2 protein, thiolase and catalase were detected in the cytosol (Figure 1C, d–f), whereas those in TKaEG1 were located in peroxisomes (Figure 1C, a–c). These import defects of peroxisomal matrix proteins in ZPEG101 were similar to those in another pex5 CHO mutant, ZP105 (Figure 1C, g–i) [10]. Although PTS1 protein import in ZPEG101 was restored by expression of PEX5L and PEX5S (Figure 1D, a–c), PTS2 import was recovered only by PEX5L (Figure 1D, d–f), as has been previously shown in ZP105 c The Authors Journal compilation c 2013 Biochemical Society 198 Figure 1 R. Natsuyama, K. Okumoto and Y. Fujiki Isolation of a peroxisome-deficient CHO cell mutant, ZPEG101 (A) Fluorescence microscopy of TkaEG1, rat PEX2 -transformed wild-type CHO-K1 stably expressing EGFP–PTS1 (a, c, e, g and i) and a peroxisome biogenesis-defective mutant ZPEG101 (b, d, f, h and j). Cells were immunostained with anti-Pex14p antibody (c and d) and were monitored by EGFP fluorescence with that of EGFP–PTS1 (a and b) using confocal microscopy. Merged views are shown in (e) and (f). Images of dual-immunostaining with antibodies to PMP70 (g and h) and Pex14p (i and j) were also shown. Scale bar, 10 μm. (B) CG analysis was done by PEX transfection. ZPEG101 was transfected with PEX1 (a), PEX5L (b), PEX14 (c) and a mock plasmid (d). At 24 h after the transfection, fluorescence of EGFP–PTS1 input was assessed as in (A). Scale bar, 10 μm. (C) Intracellular localization of peroxisomal matrix proteins in TKaEG1 (a–c), ZPEG101 (d–f), and ZP105 (g–i) was verified by immunostaining with antibodies against AOx (a, d and g), thiolase (b, e and h) and catalase (c, f and i). Scale bar, 10 μm. (D) Complementation assay with PEX5L and PEX5S . ZPEG101 was transfected with PEX5L (a and d), PEX5S (b and e) and a mock plasmid (c and f). At 24 h after the transfection, PTS1 AOx (a–c) and PTS2 thiolase (d–f) were verified as in (C). Scale bar, 10 μm. [10]. These results strongly suggested that ZPEG101 was a typical pex5 CHO cell mutant with import defects in both PTS1 and PTS2 proteins. ZPEG101 is a pex5 mutant with distinct phenotypes that has no detectable Pex5p and instable Pex14p To investigate the primary defect of ZPEG101, PEX5 cDNA was isolated by RT–PCR using the mRNA from ZPEG101 and PEX5specific primers. Subsequent sequencing of nine independent ZPEG101-derived PEX5 cDNA clones showed that a singlebase insertion of cytosine into a four cytosine tandem repeat c The Authors Journal compilation c 2013 Biochemical Society at nucleotide positions 217–220 (the A of the initiating codon ATG being base 1), resulted in a frameshift in a codon encoding Leu74 (CTT) to a proline (CCT) and creation of a premature termination codon at amino acid position 86 (Figures 2A and 2B), thereby indicating a homozygote mutation. Therefore PEX5 in ZPEG101, named PEX5ZPEG101 , is more likely to encode a shortened Pex5p with a length of 85 amino acids comprising the N-terminal 73-amino-acids of Pex5p and an additional 12amino-acid oligopeptide (Figure 2B). Transfection of HA-tagged PEX5ZPEG101 (PEX5ZPEG101 –HA) into ZPEG101 did not complement the phenotype of ZPEG101 (Figure 2C, b) as in the mocktransfected cells (Figure 2C, c), where Pex5pZPEG101 –HA was not A novel CHO mutant lacking Pex5p Figure 2 Mutation analysis of PEX5 in ZPEG101 (A) RT–PCR was performed using ClPEX5 -specific primers to amplify PEX5 derived from CHO-K1 (left-hand panel) and ZPEG101 (right-hand panel). Partial nucleotide and the deduced amino-acid sequences of respective PEX5 cDNA are shown. Nine independent PCR products of PEX5 cDNA isolated from ZPEG101 possessed a same frame-shift, one-base insertion of cytosine in a four cytosine tandem repeat from nucleotides 217–220 (italic), resulted in a frame-shift giving rise to unrelated polypeptide sequence downstream of Pro74 (bold). (B) Deduced amino-acid sequence of Pex5p derived from CHO-K1 and ZPEG101. The identical residues between normal and mutant sequences are highlighted. In ZPEG101-derived Pex5p, a double underline indicates an additional 12-amino-acid sequence created by the frameshift mutation in the codon for Leu74 (arrowhead). *, termination codon. (C) Pex5pZPEG101 showed no complementary activity in ZPEG101 cells. His-ClPEX5L–HA (a), His-ClPEX5ZPEG101 –HA (b) and a mock plasmid (c) were transfected into ZPEG101 and analysed as Figure 1(B). Scale bar, 10 μm. detectable at the expected molecular mass (Supplementary Figure S1 at http://www.biochemj.org/bj/449/bj4490195add.htm). Taken together, we concluded that primary defect of ZPEG101 is the PEX5-inactivating mutation. Next, we examined the endogenous Pex5p expression in ZPEG101 and other PEX5-deficient CHO mutants by immunoblot analysis. A small amount of Pex5p was found in a pex5 mutant ZP139 (Figure 3A, lane 3), which harbours a G522E mutation in the sixth TPR in Pex5pL (G485E in Pex5pS) [10], in contrast to the level of Pex5p in CHO-K1 (Figure 3A, lane 1). Another pex5 mutant, ZP105, showed a much lower, but detectable, level of Pex5p (Figure 3A, lane 2) with a G335E mutation (G298E in Pex5pS) in the first TPR motif [10]. These results show that a small, but significant, level of Pex5p, with a missense mutation in the TPR motifs, is expressed in these two pex5 mutants. In contrast, in ZPEG101 no discernible Pex5p (Figure 3A, lane 4) and endogenous Pex5pZPEG101 (results not shown) was observed. Given the finding that even ectopically expressed Pex5pZPEG101 – HA in ZPEG101 was hardly detectable (Supplementary Figure S1), thereby suggesting that PEX5ZPEG101 encodes a highly unstable Pex5p fragment with no complementing activity. Consistent with the morphological analysis (Figure 1D), the 52 kDa Bcomponent of PTS1 protein, AOx, which is derived from the 199 75 kDa A-component [6], and cleavage of the PTS2 signal peptide of thiolase in peroxisomes [7,8] were not detectable in ZPEG101 (Figure 3A, lane 4). This was the same for ZP105 and ZP139 (Figure 3A, lanes 2 and 3), in contrast with the normal import of these proteins in the wild-type CHO-K1 (Figure 3A, lane 1). Taken together, these results suggested that ZPEG101 is a novel pex5 CHO mutant completely lacking Pex5p protein. Moreover, in ZPEG101, Pex14p was detected at a remarkably lower level (Figure 3A, lane 4) as compared with that in CHO-K1 and ZP105 and ZP139 (Figure 3A, lanes 1–3), consistent with the barely detectable immunostaining of Pex14p (Figure 1A, d). The level of Pex13p was slightly elevated in all three of these pex5 mutants than in CHO-K1 (Figure 3A), as has been observed previously in other peroxisome matrix protein import-deficient CHO mutants [10]. Pex14p, but not Pex13p, was likewise severely reduced in HeLa cells that had been treated with PEX5 siRNA. This siRNA treatment depleted Pex5p to a barely detectable level, giving rise to severely abrogated peroxisomal import of AOx as shown by the low level of the B-component of AOx (Figure 3B). PEX14 mRNA was expressed at a normal level in ZPEG101, as in CHO-K1 (Figure 3C). No mutation was identified in PEX14 cDNA derived from ZPEG101 (results not shown), indicating that Pex14p in ZPEG101 is unstable at the protein, but not the transcriptional, level. We therefore interpreted these results to mean that the elimination of Pex5p results in a severe reduction in Pex14p. To further examine whether ectopic PEX5 expression restores such defects in ZPEG101, we established a stable transformant of ZPEG101 expressing FLAG–Pex5pL, named ZPEG101/FLPEX5L. In the immunostaining analysis of ZPEG101/FL-PEX5L cells, AOx (Figure 4A, a), as well as EGFP–PTS1, thiolase and catalase (results not shown) were colocalized with PMP70positive punctate structures (peroxisomes; Figure 4A, b) as in the parental TkaEG1 cells (Figures 1A and 1C), whereas the soluble proteins were present in the cytosol in ZPEG101 (Figure 4A, c). Immunoblot analysis also showed that the impaired import of AOx in ZPEG101 (Figure 4B, lanes 7 and 9) were restored by Pex5pL expression as verified by the appearance of the AOx component-B in the post-nuclear supernatant and organelle fractions of ZPEG101/FL-PEX5L (Figure 4B, lanes 4 and 6) as in CHO-K1 (Figure 4B, lanes 1 and 3). In addition, the observed lower level of Pex14p in ZPEG101 was restored in ZPEG101/FL-PEX5L (Figure 4B, lanes 4 and 6) to the same level as CHO-K1 (Figure 4B, lanes 1 and 3). Two RING peroxins, Pex10p and Pex2p, a membrane peroxin, Pex3p, and a major PMP, PMP70, were detected at nearly the same level in the organelle fractions from CHO-K1, ZPEG101 and ZPEG101/FLPEX5L (Figure 4C), whereas Pex12p appeared to be slightly less expressed in ZPEG101 (Figure 4C, lane 2). Together with the morphological findings (Figure 1A), these results strongly suggested that Pex14p was specifically lowered in ZPEG101. ZPEG101/FL-PEX5L was indistinguishable from CHO-K1 in the morphological and biochemical properties, although FLAG– Pex5pL was expressed in ZPEG101/FL-PEX5L cells at a higher level (∼ 10-fold) than endogenous Pex5p in CHO-K1 (Figure 4B, lanes 1–6). Therefore, we concluded that PEX5L complements peroxisomal defects of Pex14p stability as well as matrix protein import in ZPEG101. Biogenesis of Pex14p in ZPEG101 To further verify the biogenesis of Pex14p in ZPEG101, CHO-K1 and pex5 ZPEG101 were pulse-labelled for 1 h with [35 S]methionine and [35 S]cysteine and the radioactivity was chased in a medium containing no labelled amino acids. After the c The Authors Journal compilation c 2013 Biochemical Society 200 Figure 3 R. Natsuyama, K. Okumoto and Y. Fujiki Pex5p-deficient ZPEG101 shows a lower level of Pex14p (A) Wild-type CHO-K1 (lane 1) and the pex5 CHO mutants ZP105 (lane 2), ZP139 (lane 3) and ZPEG101 (lane 4) were lysed in sample buffer and analysed by SDS/PAGE. Immunoblotting was done using antibodies against the proteins indicated on the left-hand side. Note that ZPEG101 (completely lacking Pex5p) showed a lesser amount of Pex14p as compared with the other pex5 mutants, ZP105 and ZP139, in which their respective mutant forms of Pex5p were partially expressed. (B) Knockdown of Pex5p in HeLa cells decreases Pex14p protein level. HeLa cells treated for 96 h with a control siRNA (lane 1) and PEX5 siRNA (lane 2) were analysed as in (A) by immunoblotting with the antibodies indicated on the left-hand side. (C) Expression of PEX14 mRNA in ZPEG101. Northern blot analysis using mRNA (5 μg) isolated from CHO-K1 (lane 1) and ZPEG101 (lane 2) was done with 32 P-labelled probes specific against PEX14 (upper panel) and actin (lower panel). initial 1-h pulse-labelling, a similar amount of [35 S]Pex14p was detected in the immunoprecipitates from CHO-K1 and ZPEG101 with the anti-Pex14p antibody (Figure 5A, lanes 1 and 5), indicating normal synthesis of Pex14p in ZPEG101. In ZPEG101, [35 S]Pex14p was significantly reduced after a 1-h chase and barely detectable after a 2- and 6-h chase (Figure 5A, lanes 6–8), whereas [35 S]Pex14p was stable at least after a 6-h chase in CHO-K1 (Figure 5A, lanes 2–4). These results strongly suggested a rapid turnover of Pex14p in ZPEG101. PMPs including Pex14p are synthesized in free ribosomes in the cytosol, recognized by the cytosolic PMP receptor Pex19p and then transported to peroxisomes [30,31]. To examine whether the loss of Pex5p affects the translocation process of Pex14p to the peroxisomes, CHO-K1, pex5 ZPEG101 and pex14 ZP110, devoid of expression of Pex14p [21], were pulse-labelled with 35 S-labelled methionine and -cysteine for 1 h and fractionated into organelle and cytosol fractions. The immunoblot analysis showed that a normal and a small amount of Pex14p was immunoprecipitated only from the organelle fractions of CHO-K1 and ZPEG101 respectively (Figure 5B, lanes 3–6), consistent with the subcellular localization of Pex14p (Figure 4B), whereas no Pex14p was detected in both fractions of ZP110 (Figure 5B, lanes 1 and 2). On the other hand, newly synthesized [35 S]Pex14p was recovered in both cytosol and organelle fractions from ZPEG101 (Figure 5B, lanes 5 and 6), whereas [35 S]Pex14p was mostly in organelle fraction from CHO-K1 (Figure 5B, lanes 3 and 4). Collectively, these results strongly suggested that Pex14p synthesized in the cytosol is rapidly localized to peroxisomes in the wild-type CHO-K1 cells. In contrast, it is more probable that in ZPEG101 Pex14p is unstable presumably due to inefficient translocation to c The Authors Journal compilation c 2013 Biochemical Society peroxisomes, resulting in the apparent increase in its cytosolic localization and elevated susceptibility to degradation. Although Pex5p is present mainly in the cytosol, it is partly localized in the peroxisomal membrane in a proteaseresistant form, which constitutes a putative translocation complex including Pex14p and Pex13p [16,18,32,33]. To examine the possibility that peroxisome-localized Pex5p has a role in the stability of Pex14p, the organelle fractions of CHO-K1 and ZPEG101 were treated with various concentrations of NaCl and then centrifuged. In CHO-K1, the amount of Pex14p in the re-isolated organelle fraction was not altered by treatment with any of the concentrations of NaCl tested (Figure 5C, lanes 1– 5). On the other hand, after NaCl treatment less Pex14p was recovered from the ZPEG101-derived organelle fraction in a concentration-dependent manner (Figure 5C, lanes 6–10). Taken together, these results strongly suggest that Pex5p is required for the translocation, integration and stabilization of Pex14p into the peroxisome membrane. The fifth pentapeptide motif in Pex5pL is essential for Pex14p stability In the interaction of Pex5p with Pex14p, all of the seven pentapeptide WXXXF/Y motifs in the N-terminal region of Pex5pL are suggested to be binding sites for Pex14p [11,12]. The motifs 1, 3 and 5 bind to Pex14p with a much higher affinity as compared with the others [11,12,34]. To investigate which WXXXF/Y motif, if any, of Pex5pL is involved in the stabilization, we constructed a series of C-terminally HA-tagged full-length Pex5pL (Pex5pL–HA) mutants with various alanine A novel CHO mutant lacking Pex5p Figure 4 201 Expression of Pex5p restores the impaired phenotype of ZPEG101 (A) ZPEG101/FL-PEX5L, a stable formant of ZPEG101 expressing FL-ClPEX5L (a and b) and ZPEG101 (c and d) were dual immunostained with antibodies against AOx (a and d) and PMP70 (b and e). Scale bar, 10 μm. (B) Post-nuclear supernatant fractions (P) prepared from CHO-K1 (lanes 1–3), ZPEG101/FL-PEX5L (lanes 4–6) and ZPEG101 (lanes 7–9) were separated into cytosol (C) and organelle (O) fractions by ultracentrifugation. Equal aliquots of respective fractions were analysed by SDS/PAGE and immunoblotting using antibodies against the proteins indicated on the left-hand side. MDH, a mitochondrial protein. (C) Organelle fractions were prepared from CHO-K1 (lane 1), ZPEG101 (lane 2) and ZPEG101/FL-PEX5L (lane 3) as in (B). Equal aliquots were analysed by SDS/PAGE and immunoblotting with antibodies against the proteins indicated on the left-hand side. substitutions in the conserved amino acids at the positions 1 (W) and 5 (F or Y) of the seven WXXXF/Y motifs (Figure 6A). First, the respective pentapeptide-motif mutants of Pex5pL– HA were verified for binding to Pex14p. Immunoprecipitation analysis of Pex5pL–HA variants expressed in CHO-K1 cells was performed. Mut123, a Pex5pL–HA variant with mutations in the WXXXF/Y motifs 1, 2 and 3, was co-immunoprecipitated with endogenous Pex14p at a level 10 % that of the wild-type (Figure 6B, lanes 6 and 7). This is in agreement with previous studies that the Mut123 variant of the N-terminal 243 aminoacid-long Pex5pL, termed Pex5p(1-243), showed no binding to Pex14p in vitro [12]. Pex14p in the immunoprecipitate of the WXXXF/Y motif 5 mutant of Pex5pL–HA, Mut5, was moderately decreased as compared with the wild-type (Figure 6B, lane 8), whereas Pex14p was not co-immunoprecipitated with Mut1-7 (a variant with mutations in all of the seven WXXXF/Y motifs; Figure 6B, lane 9). All of the Pex5pL–HA mutants active in binding to Pex14p, including Mut123 and Mut5, were targeted to the peroxisomes in CHO-K1 (Supplementary Figure S2 at http://www.biochemj.org/bj/449/bj4490195add.htm). These results strongly suggested that the WXXXF/Y motif 5 functions as an auxiliary binding site for Pex14p in vivo, whereas Pex5pL interacts with Pex14p mainly via WXXXF/Y motifs 1–3. Furthermore, the impaired PTS1 protein import in ZPEG101 was not restored by expression of Mut123, Mut234, Mut1235, Mut1-7 or mock transfection (Figure 6C, b, c and f–h). The peroxisomal localization of EGFP–PTS1 in ZPEG101 was restored only with Mut5 and Mut567 (Figure 6C, d and e) to the level of wild-type Pex5pL–HA (Figure 6C, a). Taken together, these results suggest that the binding of Pex5pL to Pex14p via WXXXF/Y motifs 1–3 was necessary for the complementing activity and that WXXXF/Y motif 5-mediated binding was dispensable (Figure 6D, bottom panel). Next, the Pex5pL–HA mutants were examined to investigate whether they restore the reduced level of Pex14p in ZPEG101. When full-length Pex5pL–HA was transiently expressed in ZPEG101, the level of Pex14p was elevated (Supplementary Figure S3, lane 1 at http://www.biochemj.org/bj/449/bj4490195add. htm) as was also observed in ZPEG101 stably expressing FL-Pex5pL (Figure 4B). Pex14p in ZPEG101 was less, but significantly, increased by expression of Pex5pL(1-305) containing all seven of the WXXXF/Y motifs (Supplementary Figure S3, lane 2), whereas Pex5pL(1-243), harbouring WXXXF/Y motifs 1–4, and Pex5pL(305-632) did not restore the levels of Pex14p (Supplementary Figure S3, lanes 3 and 4), as in the mock transfection (Supplementary Figure S3, lane 5). These results suggest that the N-terminal region of Pex5pL containing the WXXXF/Y motifs, especially motifs 5–7, plays an important role in stabilizing the level of expressed Pex14p in ZPEG101. The expression of Mut234, a Pex5pL–HA mutant with normal c The Authors Journal compilation c 2013 Biochemical Society 202 Figure 5 R. Natsuyama, K. Okumoto and Y. Fujiki Biogenesis of Pex14p in ZPEG101 (A) CHO-K1 (lanes 1–4) and ZPEG101 (lanes 5–8) were labelled with [35 S]methionine and [35 S]cysteine for 1 h and were chased for the indicated times at the top. The cells were lysed and subjected to immunoprecipitation with an anti-Pex14p antibody. Upper panel, the immunoprecipitates and the total cell lysates (5 %) were separated by SDS/PAGE and analysed by autoradiography for [35 S]Pex14p and immunoblotting with an anti-LDH antibody. Lower panel, [35 S]Pex14p level at each time point was quantified, normalized with LDH as a loading control and plotted by taking as 1 the level at 0 h chase. Results are means + − S.E.M. for three independent experiments. *P < 0.05 and **P < 0.01. (B) pex14 ZP110 (lanes 1 and 2), CHO-K1 (lanes 3 and 4) and pex5 ZPEG101 (lanes 5 and 6) cells were pulse-labelled with [35 S]methionine and [35 S]cysteine as in (A) and fractionated as described in the Materials and methods section. Immunoprecipitated Pex14p from equal aliquots of the resultant supernatant (S) and pellet (P) fractions was analysed by autoradiography (top panel) and immunoblotting with anti-Pex14p antibody (upper middle panel). Equal aliquots of the supernatant and pellet fractions from the respective cells were analysed by SDS/PAGE and immunoblotting using antibodies against an endoplasmic membrane protein, cytochrome P450 reductase (P450R; lower middle panel) and cytosolic LDH (bottom panel). (C) Integrity of Pex14p in ZPEG101. Upper panel, organelle fractions of CHO-K1 (lanes 1–4) and ZPEG101 (lanes 5–8), prepared as in (B), were incubated for 30 min on ice in buffer containing various concentrations of NaCl as indicated at the top. Reaction mixtures were centrifuged. Pellet fractions were analysed by immunoblotting with antibodies to Pex14p and Pex13p. Lower panel, the Pex14p level was normalized with Pex13p as a loading control. Results are means + − S.E.M. for three independent experiments. **P < 0.01. binding to Pex14p, but deficient in the binding to Pex13p [12], restored the level Pex14p in ZPEG101, nearly to the same level as observed in CHO-K1 (Figure 6D, lanes CHO-K1 and Mut234) and the wild-type Pex5pL–HA (Figure 6D, lane WT). The Pex14p level in ZPEG101 was likewise restored by expression of Mut123 (Figure 6D, lane Mut123), despite its lower binding activity to Pex14p (Figure 6B, lane 7), thereby suggesting that binding of Pex5pL to Pex13p and Pex14p via WXXXF/Y motifs 1–3 was dispensable for sustaining the stability of Pex14p. In contrast, Mut5 and other multiple WXXXF/Y-motif mutants containing the motif 5 mutation, showed only a slightly elevated level of Pex14p (Figure 6D, lanes Mut5, Mut567, Mut1235 and Mut1-7), suggesting that the WXXXF/Y motif 5, not motifs 1–4, is essential for the Pex14p stability. Taken together, the results suggest that the WXXXF/Y motifs 1 and 3 of Pex5pL mainly function in the binding to Pex14p and the matrix protein import activity, whereas the WXXXF/Y motif 5 has an essential role in the stabilizing Pex14p in the peroxisome membrane (Figure 6D, bottom panel). Next, we expressed wild-type Pex5pS–HA, a shorter form of Pex5p lacking a Pex5pL-specific 37 amino-acid insertion containing the WXXXF/Y motif 5, in ZPEG101. Pex5pS–HA restored the reduced level of Pex14p in ZPEG101, but less c The Authors Journal compilation c 2013 Biochemical Society efficiently than Pex5pL–HA (Figure 6E, lanes 2 and 4) in a manner dependent on the WXXXF/Y motifs excluding motif 5 where no restoration of the lowered Pex14p was observed with Mut1-4,6,7 (all of the six WXXXF/Y motifs in Pex5pS were mutated, where the fifth and sixth WXXXF/Y motifs in Pex5pS are numbered as 6 and 7 respectively; Figure 6E, lane 5). Together with the results obtained with various WXXXF/Y motif mutants of Pex5pS–HA, we conclude that the motifs 1 and 7 possess the Pex14p-stabilizing activity in Pex5pS, with a relatively higher efficiency of motif 1 as compared with motif 7 (Figure 6E). Thus the WXXXF/Y motifs 1 and 7 of Pex5pS are more likely to play a compensatory role for the function of motif 5 of Pex5pL. Pex5p–Pex13p interaction is essential for the import of PTS1 proteins and catalase, but not for PTS2 proteins On the basis of an assay with a Pex5pL mutant, Mut234, in the pex5 mutant ZP105, we earlier suggested that interactions between Pex5pL and Pex13p are required for peroxisomal import of catalase, but not PTS1 and PTS2, in mammalian cells [12]. However, ZP105 might not be the most suitable mutant to evaluate A novel CHO mutant lacking Pex5p Figure 6 203 Pentapeptide WXXXF/Y motif 5 in Pex5pL is essential for stabilization of Pex14p (A) Schematic representation of the domain structure of Chinese hamster Pex5pL and Pex5pS. Seven pentapeptide (WXXXF/Y)-motifs (solid horizontal bars), a 37-amino-acid insertion (grey box) and a C-terminal region containing seven TPR motifs (black box) are shown in Pex5pL, whereas six WXXXF/Y motifs lacking the fifth one are shown in Pex5pS. (B) Interaction of Pex5p with Pex14p via WXXXF/Y motifs. CHO-K1 cells transfected with wild-type PEX5L–HA and its variants were immunoprecipitated (IP) with anti-HA antibody (lanes 6–10) and analysed by immunoblotting using antibodies against HA and Pex14p. Input (5 %) was loaded in lanes 1–5. (C) Wild-type PEX5L–HA and its variants were transfected into ZPEG101 and their complementing activity of ZPEG101 was verified by import of EGFP–PTS1 as in Figure 1(A). Scale bar, 10 μm. (D) Restoration of Pex14p protein level in ZPEG101 cells. ZPEG101 cells were transfected with the wild-type PEX5L–HA and its variants and analysed by immunoblotting as in (B). LDH was detected as a loading control. Wild-type Pex5pL–HA (WT) and the variants were verified for restoring PTS1 protein import activity (PTS1 import) and Pex14p level (stability) in ZPEG101 and for their localization into peroxisomes (Ps localization) in CHO-K1 cells (bottom panels). − , negative; + , positive; + + , highly positive. (E) Involvement of Pex5pS in Pex14p stability. Pex5pS, a shorter form of Pex5p, lacks a Pex5pL-specific 37-amino-acid insertion containing the WXXXF/Y motif 5 (see a schematic view in A). The fifth and sixth WXXXF/Y motifs in Pex5pS are numbered as motifs 6 and 7 respectively, because these are the motifs equivalent to the sixth and seventh ones in Pex5pL. ZPEG101 cells were transfected with the wild-type PEX5S–HA and its variants and analysed by immunoblotting as in (D). Note that the wild-type PEX5S-HA restored the lowered Pex14p level in ZPEG101 cells, but less efficiently than wild-type PEX5L–HA (lanes 2 and 4). This Pex14p-stabilizing activity of Pex5pS was reduced largely in Mut1-4 and Mut1 (lanes 6 and 9), partially in Mut6,7 and Mut7 (lanes 7 and 10), and almost completely in Mut1,7 and Mut1-4,6,7 (lanes 8 and 5). Mut2 and Mut3 showed full activity and were as active as the wild-type (lanes 4, 11 and 12). the complementary activity of Pex5p variants because a very low level of the Pex5p mutant with a missense mutation in the first TPR motif [12] is expressed at 37 ◦ C in ZP105 (Figure 3A, lane 2), with a temperature-sensitive phenotype expressing a normal level of Pex5p [13]. Therefore we also verified the importance of Pex5p–Pex13p interaction in matrix protein import in pex5 ZPEG101 without Pex5p. Wild-type Pex5pL–HA and the Mut234 mutant were separately expressed in ZP105 or ZPEG101 and the import of PMPs was analysed by immunofluorescent microscopy and immunoblotting. In ZP105, peroxisomal import of the PTS1 protein AOx and the PTS2 protein thiolase was restored with both the wildtype Pex5pL and Mut234, whereas catalase import was reestablished only with the wild-type Pex5pL (Supplementary Figure S4 at http://www.biochemj.org/bj/449/bj4490195add.htm) as reported previously [12]. Upon expression of Mut234 in ZP105, peroxisomal AOx import was also biochemically assessed by conversion of the A-polypeptide chain of AOx into the B-chain [6]. This occurred with less efficiency as compared with the wild- type Pex5pL–HA (Figure 7A, lanes 1 and 2). Under the same experimental conditions, upon Mut234 expression in ZPEG101, the impaired import of thiolase was increased (Figure 7B, h), but catalase was not imported (Figure 7B, b), as observed in ZP105 (Supplementary Figure S4, b and h), whereas the import of both proteins was restored with wild-type Pex5pL–HA (Figure 7B, a and g). After expression of Mut234 in ZPEG101, AOx was stained in a diffuse pattern in the cytosol (Figure 7B, e), where the AOx B-polypeptide chain was not detectable (Figure 7A, lane 5). The lower level of Pex14p in ZPEG101 was altered to a normal level comparable with wild-type Pex5pL by Mut234 expression (Figure 7A, lanes 4–6), consistent with the data shown in Figure 6(D). These results strongly suggested that in ZPEG101 Mut234 was defective in not only the impaired import of catalase, but also PTS1 proteins. In the immunoprecipitation assay, wild-type Pex5pL–HA, but not Mut234, was observed to co-immunoprecipitate with FLAG–Pex13p expressed in CHOK1 cells (Figure 7C, lanes 1–6), whereas both the wild-type Pex5pL–HA and Mut234 were equally co-immunoprecipitated c The Authors Journal compilation c 2013 Biochemical Society 204 Figure 7 R. Natsuyama, K. Okumoto and Y. Fujiki Pex5p–Pex13p interaction is required for import of PTS1 protein, but not for PTS2 protein (A) Pex5pL(Mut234) restores Pex14p level, but does not restore the import defect of AOx, a PTS1 protein in ZPEG101. pex5 ZP105 (lanes 1–3) and pex5 ZPEG101 cells (lanes 4–6) were transfected with the wild-type PEX5L (WT)–HA and PEX5L(Mut234)–HA and analysed by immunoblotting with the antibodies indicated on the left-hand side. (B) Pex5pL(Mut234) complements the import defects of PTS2 protein, not PTS1 protein and catalase, in ZPEG101. ZPEG101 cells were transfected with PEX5L (WT)–HA (a, d and g), PEX5L(Mut234)–HA (b, e and h), and a mock plasmid (c, f and i), and immunostained with antibodies against catalase (a–c), AOx (d–f) and thiolase (g–i) as in Figure 1(A). Scale bar, 10 μm. (C) Interaction of Pex5pL with Pex13p and Pex14p. FL-PEX13 and FL-PEX14 were co-transfected into 207P7 cells with PEX5L(WT)–HA , PEX5L(Mut234)–HA and a mock plasmid. Cells were analysed by immunoprecipitation (IP) with anti-FLAG IgG-agarose (lanes 4–6 and 10–12) and by immunoblotting using antibodies against HA and FLAG. Input (5 %) was loaded in lanes 1–3 and 7–9. c The Authors Journal compilation c 2013 Biochemical Society A novel CHO mutant lacking Pex5p with FLAG–Pex14p (Figure 7C, lanes 7–12). These results suggest that mutations in WXXXF/Y motif 2–4 abrogated the interaction of Pex5p with Pex13p, but not with Pex14p in vivo, in a good agreement with the findings by pull-down assay using the GST (glutathione transferase) fusion protein of Mut234 [12]. Taken together, re-verifying Mut234 in ZPEG101 absent from Pex5p revealed that the Pex5p–Pex13p interaction is essential for import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. DISCUSSION In the present study, we isolated another pex5 CHO mutant, ZPEG101, defective in the import of both PTS1 and PTS2 proteins like ZP105 [10]. ZPEG101 is distinct from other known pex5 mutants in the level of its Pex5p expression; Pex5p is not detectable and the level of its receptor Pex14p is severely reduced (Figure 3A). In ZPEG101, a homozygous frame-shift mutation of PEX5 gives rise to an 85-amino-acid long premature, and apparently unstable, Pex5p (Figure 2 and Supplementary Figure S1). In ZP105 and ZP139, low, but significant, amounts of Pex5p, which harbour a missense mutation in the first and sixth TPR motifs respectively [10], are discernible (Figure 3A). Pex5p mutants lacking the C-terminal region such as Pex5p(1-243) are expressed at the same level as the wild-type Pex5p and are indeed sufficient for PTS2 protein import [12]. Accordingly, it is more probable that a lower level of Pex5p expression in ZP105 and ZP139 is sufficient for sustaining Pex14p stability. Therefore ZPEG101 is the first pex5 CHO mutant that shows a barely detectable Pex14p level and no detectable Pex5p. In addition to the complementarity in the defects in PMP import in ZPEG101, the lower protein level of Pex14p is restored by Pex5pL expression (Figures 4B and 6D), clearly indicating that Pex5p is responsible for the Pex14p stability in peroxisomes. Pex5pL interacts with Pex14p via seven WXXXF/Y motifs [11,12]. In the present study using various Pex5pL mutants, the WXXXF/Y motifs 1–4 are dispensable for stabilizing Pex14p in ZPEG101 (Figure 6D), whereas these motifs are the main binding sites to Pex14p in vitro and are required for the complementation of the impaired matrix protein import (Figure 6C) [12]. Moreover, to maintain a normal level of Pex14p, the WXXXF/Y motif 5 of Pex5pL is also essential (Figure 6D), which is another strong Pex14p-binding site with unknown function [11,12]. Pex5pS, devoid of a Pex5pL-specific WXXXF/Y motif 5-containing insertion, likewise shows less, but significant, Pex14p-stabilizing activity via WXXXF/Y motifs 1 and 7 (Figure 6E). We interpret these results to mean that Pex14p stability, predominantly sustained by the WXXXF/Y motif 5 of Pex5pL, is compensated for by binding to Pex5pS via WXXXF/Y motifs 1 and 7. This is in good agreement with the finding that the level of Pex14p is not affected in another recently isolated pex5 CHO mutant, ZPEG241, which expresses Pex5pS and not Pex5pL [35]. Thus these data clarify the novel function of Pex5pL via the WXXXF/Y motif 5 in mammalian cells, in addition to the essential role of WXXXF/Y motifs 1– 3 in the complementing activity (Figures 6C and 6D). Pex5pLspecific insertion is required for the binding of Pex5pL to Pex7p and the peroxisomal import of PTS2 proteins [10,13]. Therefore it is probable that Pex5pL forms a WXXXF/Y motif 5-mediated more stable complex with Pex14p during the translocation of PTS2 proteins, whereas the Pex5pS–Pex14p complex is distinctly and transiently assembled during the import of PTS1 proteins. Moreover, Pex14p level may be reduced in PEX5-knockout mice [36], similar to PEX5-depleted HeLa cells (Figure 3B) and 205 ZPEG101 (Figure 3A). The number of WXXXF/Y motifs and the function are varied in species (for a review see [37]). For example, a reduced level of Pex14p was not observed in pex5null mutant Saccharomyces cerevisiae [38]. S. cerevisiae Pex5p possesses two distinct binding sites for Pex14p: the inverted WXXXF motif for the Pex14p-N region [39] and another potential site that has not been fully elucidated for the Pex14p-C region [40,41]. The mammal-specific instability of Pex14p in Pex5pdeficient cells is attributed to the WXXXF/Y motif-dependent Pex5p–Pex14p interaction, which is different from the mode of interaction in yeast ([11,12], and the present study). Alternatively, in pex5-null yeast mutants Pex14p may be anchored to the membrane and stabilized by Pex8p and Pex17p, two yeastspecific peroxins that interact with Pex14p in peroxisomes [16]. Therefore WXXXF/Y motifs 1–4 in mammalian Pex5pL are likely to be a core element essential for peroxisomal protein import and WXXXF/Y motifs 5–7 confer additional functions such as maintaining Pex14p stability, which is acquired during the evolution. Indeed WXXXF/Y motif 5 was not essential for the restoration of PMP import (Figure 6C) and its targeting to peroxisomes (Supplementary Figure S2, g and h). Consistent with our notion, it is noteworthy that the WXXXF/Y motif 6 of human Pex5pL was recently shown to have an important role in the import of catalase into peroxisomes [42]. How Pex5pL regulates the level of Pex14p remains to be defined. Almost all PMPs including Pex14p are imported by the Pex19p-dependent Class I pathway, where the PMP receptor Pex19p binds PMPs in the cytosol and translocates them into peroxisomes [30,31]. In Pex5p-deficient ZPEG101 cells, about 50 % of the newly synthesized Pex14p is localized in peroxisomes with the remainder apparently residing in the cytosol, in contrast with Pex14p which is exclusively localized to the peroxisomes in CHO-K1 cells (Figure 5B). Moreover, Pex14p localized to the peroxisomes in ZPEG101 is more sensitive to the high-salt wash compared with Pex14p in CHO-K1 (Figure 5C). These results suggest that in ZPEG101 the less efficient integration of Pex5p into the peroxisomal membrane or Pex14p-containing membrane protein complexes [33,43] leads to a lower level of Pex14p. In the shuttling of Pex5p between peroxisomes and the cytosol in normal mammalian cells, Pex5p initially forms ∼ 800-kDa complexes with Pex14p and Pex13p upon peroxisomal import [18]. In yeast, an ion-conducting channel activity was recently reported on a similar complex mainly consisting of Pex5p and Pex14p in the peroxisomal membrane, implicating a peroxisomal import pore [44]. Therefore it is more probable that peroxisomelocalized Pex5p functions in maintaining the level of Pex14p via the WXXXF/Y motif 5. Otera et al. [12] reported that the interaction of Pex5pL with the N-terminal region of Pex13p via WXXXF/Y motifs 2–4 was required for the import of catalase, but not for that of PTS1 and PTS2 proteins in mammalian cells, on the basis that the Mut234 mutant of Pex5pL, defective in the interaction with Pex13p, restored the impaired import of PTS1 and PTS2 proteins and not catalase in ZP105. In the present study, we re-evaluated Mut234 with the Pex5p-less mutant cell line ZPEG101. The import defect of PTS1 proteins as well as catalase was not restored by Mut234 expression in ZPEG101 (Figures 7A and 7B), thereby suggesting an essential role of the Pex5p–Pex13p interaction in the import of PTS1 protein and catalase. A present explanation for the different phenotype of Mut234 in the two pex5 mutants ZP105 and ZPEG101 may include the presence of a mutated form of Pex5p at a low level in ZP105, in contrast with no discernible Pex5p in ZPEG101 (Figure 3A). It is more likely to be in ZP105 that a small amount of endogenous Pex5p harbouring a mutation in the TPR motif 1 [10] and exogenously expressed Mut234 c The Authors Journal compilation c 2013 Biochemical Society 206 R. Natsuyama, K. Okumoto and Y. Fujiki mutually complement their respective defect in the cargo protein import by forming a hetero-oligomer [13,45]. This interpretation is in good agreement with the data that Mut234 alone does not re-establish the peroxisomal import of PTS1 proteins and catalase in ZPEG101 (Figures 6C and 7B). PTS1-cargo-loaded Pex5p is initially targeted to peroxisomes via the binding to Pex14p and is translocated to the ∼ 800-kDa complexes including Pex13p [18]. Therefore interaction of Pex5p with Pex13p might play an important role in the formation of the initial translocation machinery in PTS1 protein import. In contrast with PTS1 protein import, Pex5p–Pex13p interaction is dispensable for PTS2 protein import (Figure 7B) [12]. Pex5pL specifically associates with the PTS2 receptor Pex7p and mediates the targeting of the Pex7p–PTS2 protein complex into peroxisomes by docking to Pex14p [13–15]. However, after localization of Pex7p to the peroxisomes, the interaction of Pex7p with Pex13p is not mediated by Pex5pL [15]. Therefore these reports and the results of the present study (Figure 7B) strongly suggested that after the targeting of Pex7p–PTS2 protein complex to the peroxisomes, PTS2 protein is translocated into the peroxisome matrix in a manner independent of Pex5p–Pex13p interaction. This is in good agreement with the differential import of PTS1 and PTS2 proteins in in vitro transport system [46]. A Pex14p mutant lacking affinity to Pex13p has no complementing activity for the impaired import of PTS1 and PTS2 proteins in the pex14 CHO mutant ZP161 [43], strongly suggesting that the binding of Pex13p to Pex14p, and not Pex5p, is essential for PTS2 protein import. Interaction of Pex5p with Pex13p is also conserved in S. cerevisiae [47,48] and Pichia pastoris [49]. However, yeast Pex5p binds to Pex13p at the SH3 domain [47–49] and not at the N-terminal region that is defined in mammalian cells [12]. Molecular mechanisms underlying the dynamic formation and function of complexes including Pex5p, Pex14p and Pex13p remain to be investigated in more details [50]. Pex5p-less ZPEG101 would be useful for addressing such issues. AUTHOR CONTRIBUTION Kanji Okumoto and Yukio Fujiki designed the research; Ryuichi Natsuyama and Kanji Okumoto performed the research; Ryuichi Natsuyama, Kanji Okumoto and Yukio Fujiki analysed the data; and Ryuichi Natsuyama, Kanji Okumoto and Yukio Fujiki wrote the paper. ACKNOWLEDGEMENTS We thank M. Nishi for the Figure illustrations, S. Okuno for technical assistance and the other members of our laboratory for discussions. FUNDING This work was supported, in part, by the Science and Technology Agency of Japan, Grantsin-Aid for Scientific Research [grant numbers 19058011, 20370039 and 24247038 (to Y.F.)], The Global COE Program from The Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Takeda Science Foundation and Japan Foundation for Applied Enzymology. REFERENCES 1 Wanders, R. J. A. and Waterham, H. R. (2006) Biochemistry of mammalian peroxisomes revisited. Annu. Rev. Biochem. 75, 295–332 2 Fujiki, Y., Okumoto, K., Kinoshita, N. and Ghaedi, K. 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FEBS J. 277, 3268–3278 Received 5 June 2012/24 September 2012; accepted 26 September 2012 Published as BJ Immediate Publication 26 September 2012, doi:10.1042/BJ20120911 c The Authors Journal compilation c 2013 Biochemical Society Biochem. J. (2013) 449, 195–207 (Printed in Great Britain) doi:10.1042/BJ20120911 SUPPLEMENTARY ONLINE DATA Pex5p stabilizes Pex14p: a study using a newly isolated pex5 CHO cell mutant, ZPEG101 Ryuichi NATSUYAMA*, Kanji OKUMOTO*† and Yukio FUJIKI*†1 *Graduate School of Systems Life Sciences, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, and †Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Figure S1 Pex5pZPEG101 is undetectable upon its overexpression Expression and complementation activity of Pex5pZPEG101 . ZPEG101 cells were transfected with wild-type His-PEX5L–HA (lane 1), His-PEX5LZPEG101 –HA (lane 2) or a mock plasmid (lane 3). At 24 h after transfection, cells were lysed and analysed by immunoblotting using antibodies against His (upper panel) and LDH (lower panel). Arrowhead indicates His-Pex5pL–HA. Molecular mass markers are shown on the left-hand side in kDa. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2013 Biochemical Society R. Natsuyama, K. Okumoto and Y. Fujiki Figure S2 WXXXF/Y motifs 1–3 and 5 are required for the peroxisomal localization of Pex5p ZPEG101 cells were transfected with various PEX5L–HA mutants. At 24 h after the transfection, cells were permeabilized with digitonin to remove the cytosol and dual immunostained with antibodies against HA (a, c, e, g, i, k, m and o) and PMP70 (b, d, f, h, j, l, n and p), as in Figure 4(A) of the main text. Wt, wild-type. c The Authors Journal compilation c 2013 Biochemical Society A novel CHO mutant lacking Pex5p Figure S4 Pex5pL(Mut234), a Pex5pL mutant defective in the binding to Pex13p, restores import of PTS1 and PTS2 proteins in ZP105 Figure S3 N-terminal 243 amino-acids of Pex5pL, Pex5pL(1-243), is not sufficient for stabilization of Pex14p ZPEG101 was transfected with plasmids each encoding wild-type Pex5pL-FLAG (Full length; lane 1), Pex5pL(1-305) (lane 2), Pex5pL(1-243) (lane 3) and Pex5pL(306-632) (lane 4), plus a mock plasmid (lane 5). At 24 h after the transfection, cells were lysed and analysed by immunoblotting with antibodies against Pex14p, FLAG and LDH as in Figure 3(A) of the main text. ZP105 cells transfected with wild-type PEX5L–HA (a, d and g), PEX5L(Mut234)–HA (b, e and h), and a mock plasmid (c, f and i) were immunostained with antibodies to catalase (a–c), AOx (d–f) and thiolase (g–i) as in Figure 7(B) of the main text. Note that in contrast with ZPEG101, Pex5pL(Mut234) complemented the import defects of PTS1 and PTS2 proteins in ZP105, but not catalase [1]. Scale bar, 10 μm. REFERENCE 1 Otera, H., Setoguchi, K., Hamasaki, M., Kumashiro, T., Shimizu, N. and Fujiki, Y. (2002) Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import. Mol. Cell. Biol. 22, 1639–1655 Received 5 June 2012/24 September 2012; accepted 26 September 2012 Published as BJ Immediate Publication 26 September 2012, doi:10.1042/BJ20120911 c The Authors Journal compilation c 2013 Biochemical Society