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Isolation and Characterization of Pas2p, a Peroxisomal Membrane Protein Essential for Peroxisome Biogenesis in the Methylotrophic Yeast Pichia pastoris

Isolation and Characterization of Pas2p, a Peroxisomal Membrane Protein Essential for Peroxisome... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 31, Issue of August 2, pp. 18973–18980, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Isolation and Characterization of Pas2p, a Peroxisomal Membrane Protein Essential for Peroxisome Biogenesis in the Methylotrophic Yeast Pichia pastoris* (Received for publication, April 5, 1996) Erik A. C. Wiemer‡§, Georg H. Lu¨ ers‡, Klaas Nico Faber‡, Thibaut Wenzel‡, Marten Veenhuis , and Suresh Subramani‡ From the ‡Department of Biology, University of California at San Diego, La Jolla, California 92093-0322, and the ¶Laboratory for Electron Microscopy, University of Groningen, Biological Center, 9751 NN Haren, The Netherlands The pas2 mutant of the methylotrophic yeast Pichia involvement in H O metabolism and the b-oxidation of fatty 2 2 pastoris is characterized by a deficiency in peroxisome acids (Van den Bosch et al., 1992; Wiemer and Subramani, biogenesis. We have cloned the PpPAS2 gene by func- 1994). The importance of peroxisomes is emphasized by the tional complementation and show that it encodes a pro- existence of severely debilitating, and often lethal, human dis- tein of 455 amino acids with a molecular mass of 52 kDa. eases in which peroxisome biogenesis is impaired (Lazarow and In a Pppas2 null mutant, import of both peroxisomal Moser, 1989). In fungi, peroxisomes are the sole site for b-oxi- targeting signal 1 (PTS1)- and PTS2-containing proteins dation of fatty acids and, in methylotrophic yeasts, are essen- is impaired as shown by biochemical fractionation and tial for the oxidation of methanol (Kunau et al., 1987; Veenhuis, fluorescence microscopy. No morphologically distin- 1992). Much progress has been made in recent years delineat- guishable peroxisomal structures could be detected by ing the molecular requirements for import of peroxisomal ma- electron microscopy in Pppas2 null cells induced on trix proteins (metabolic enzymes). Typically, these proteins are methanol and oleate, suggesting that PpPas2p is in- synthesized on cytosolic polysomes and imported posttransla- volved in the early stages of peroxisome biogenesis. tionally without further modifications. Two evolutionary con- PpPas2p is a peroxisomal membrane protein (PMP) and served peroxisomal targeting signals (PTSs) for matrix pro- is resistant to extraction by 1 M NaCl or alkaline sodium teins have been identified (Subramani, 1993). Most frequently carbonate, suggesting that it is a peroxisomal integral encountered is PTS1, consisting of a COOH-terminal tripeptide membrane protein. Two hydrophobic domains can be with the consensus sequence S/A/C-K/R/H/-L/M (Gould et al., distinguished which may be involved in anchoring 1989; Keller et al., 1991). PTS2 is found at the amino termini of PpPas2p to the peroxisomal membrane. PpPas2p is ho- a smaller subset of peroxisomal proteins and conforms to the mologous to the Saccharomyces cerevisiae Pas3p. The consensus RL-X -H/QL (Swinkels et al., 1991; Osumi et al., first 40 amino acids of PpPas2p, devoid of the hydropho- 1991; Erdmann, 1994; Glover et al., 1994; Faber et al., 1995). bic domains, are sufficient to target a soluble fluores- cent reporter protein to the peroxisomal membrane, Separate import pathways exist for PTS1- and PTS2-contain- with which it associates tightly. A comparison with the ing proteins since both in yeast and mammalian cells import membrane peroxisomal targeting signal of PMP47 of defects have been observed excluding either PTS1- or PTS2- Candida boidinii revealed a stretch of positively containing proteins from the peroxisome (McCollum et al., charged amino acids common to both sequences. The 1993; Motley et al., 1994; Marzioch et al., 1994; Slawecki et al., role of peroxisomal membrane targeting signals and 1995; Zhang and Lazarow, 1995). Functional complementation transmembrane domains in anchoring PMPs to the per- of the yeasts has revealed the PTS1 (PpPas8p, ScPas10p, oxisomal membrane is discussed. HpPer3p, and YlPay32p) and PTS2 receptors (ScPas7p or ScPeb1p) that specifically interact with their cognate signals (McCollum et al., 1993; Van der Leij et al., 1993; Marzioch et Peroxisomes (microbodies) comprise an inducible and versa- al., 1994; Zhang and Lazarow, 1995, 1996; Terlecky et al., tile eukaryotic subcellular compartment that plays a key role 1995). in a number of metabolic pathways. A general feature is its Besides the yeast mutants with a selective import defect for matrix proteins, a large collection of strains has been described which show a general defect in peroxisome assembly (pas, per * This work was supported in part by a grant from the Human pay,or peb mutants; Erdmann et al., 1989; Cregg et al., 1990; Frontier Science Program Organization (HFSPO) and the American Gould et al., 1992; Liu et al., 1992; Van der Leij et al., 1992; Heart Association (to E. A. C. W.), an HFSPO fellowship (to K. N. F.), a fellowship from the Deutsche Forschungsgemeinshaft (to G. L.), a fel- Elgersma et al., 1993; Nuttley et al., 1993; Zhang and Lazarow, lowship from the European Molecular Biology Organization (to T. W.), 1993). In these strains the bulk of the peroxisomal matrix and by National Institutes of Health Grant NIHDK41737 and National proteins is found in the cytosol. Some contain peroxisomes with Science Foundation Grant MCB9316018 (to S. S.). The costs of publi- an aberrant morphology reminiscent of the peroxisomal ghost cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” structures found in fibroblast cell lines derived from patients in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with peroxisomal disorders (Wiemer et al., 1989). The nucleotide sequence(s) reported in this paper has been submitted TM to the GenBank /EBI Data Bank with accession number(s) Z72390. § Present address: Institute for Hematology, Erasmus University, Rotterdam, The Netherlands. The abbreviations used are: PTS(s), peroxisomal targeting signal(s); To whom correspondence should be addressed: Dept. of Biology, PMP, peroxisomal membrane protein; mPTS, peroxisomal membrane UCSD, Rm. 4314, Bonner Hall, 9500 Gilman Dr., La Jolla, CA 92093- targeting signal; PCR, polymerase chain reaction; kb, kilobase; GFP, 0322. Tel.: 619-534-2327; Fax: 619-534-0053; E-mail: subra@ green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; jeeves.ucsd.edu. TMD, transmembrane domain; PAS, peroxisome assembly. This is an open access article under the CC BY license. 18973 18974 PpPAS2, a Gene Involved in Peroxisomal Biogenesis TABLE I P. pastoris strains Strain Genotype (expression construct) Source PPY1 21-1 Wild type Y-11430, NRRL PPY4 Pphis4 Gould et al. (1992) PPY12 Pphis4, Pparg4 Gould et al. (1992) PPY21 Pppas2, Pparg4 Gould et al. (1992) SEW1 (Pppas2 null) Pphis4, Pppas2<PpARG4 This paper SEW2 Pppas2<PpARG4, Pphis4::pTW51 (P GFP-PTS1) This paper AOXI SEW3 Pppas2<PpARG4, Pphis4<pTW66 (P PTS2-GFP) This paper GAPDH STW1 Pparg4, Pphis4<pTW51 (P GFP-PTS1) This paper AOXI STW2 Pparg4, Pphis4<pTW66 (P PTS2-GFP) This paper GAPDH SKF1 Pparg4, Pphis4<pKNSD77 (P 1-40-GFP) This paper AOXI fragment (polylinker sites) into the EcoRI site (blunted with Klenow Complementing genes have been isolated and characterized polymerase) of pJAH35, a pBR322-based vector containing the PpHIS4 for most of these mutants. Several of the encoded proteins were gene and a P. pastoris autonomously replicating sequence (PARS2, shown to be membrane-associated. The functions and molecu- Cregg et al., 1985). lar requirements for targeting of these peroxisomal membrane Plasmid pTW51, containing the green fluorescent protein-PTS1 proteins (PMPs), which do not contain a PTS1 or PTS2, are (GFP-PTS1) construct under control of the methanol-inducible alcohol largely unknown. oxidase promoter (P ), was obtained by cloning an EcoRI PCR prod- AOX1 Here we report the cloning of the Pichia pastoris PAS2 gene, uct of the GFPS65T mutant allele (Heim et al., 1995) in the P. pastoris integrating vector pHIL-D2 (Invitrogen, San Diego). The primer at the encoding a peroxisomal membrane protein (PpPas2p) which is 39 end contained the appropriate codons to include SKL (PTS1 signal) in essential for peroxisome biogenesis. The phenotype of the Pp- the predicted translation product (Monosov et al., 1996). Plasmid pas2 null strain is examined, and the peroxisomal membrane- pTW66, containing the PTS2-GFP construct under control of the con- targeting signal (mPTS) of PpPas2p is delineated. stitutive glyceraldehyde 3-phosphate dehydrogenase promoter (P ; GAPDH gift of Dr. J. M. Cregg, Oregon Graduate Institute of Science and MATERIALS AND METHODS Technology, Portland, OR) was constructed by cloning the GFPS65T Yeast Strains and Culture Conditions—The P. pastoris strains used allele in-frame with a part of the ScFOX3 sequence (Glover et al., 1994) in this study are listed in Table I. Yeast strains were grown at 30 °C in encoding the NH -terminal 16 amino acids of the Saccharomyces cer- YPD (1% w/v yeast extract, 2% w/v Bacto-peptone, 2% w/v dextrose), evisiae 3-oxoacyl-CoA thiolase in vector pHIL-D2. YPM (1% w/v yeast extract, 2% w/v Bacto-peptone, 0.5% v/v methanol), A hybrid gene encoding the NH -terminal 40 amino acids of PpPas2p YPOT (1% w/v yeast extract, 2% w/v Bacto-peptone, 0.2% v/v oleate, and GFP (1–40-GFP) was constructed as follows. By PCR, an Asp-718 0.02% v/v Tween 40), or in synthetic medium consisting of 0.67% w/v and a BglII site were introduced upstream of the PpPAS2 open reading yeast nitrogen base, supplemented with 50 mg/ml of the appropriate frame present in pBSIIKS, resulting in pKNSD44. A second PCR was amino acids and with one of the following carbon sources: 2% w/v performed to amplify a DNA fragment encoding the GFPS65T allele dextrose (SD), 0.5% v/v methanol (SM), or 0.2% v/v oleate and 0.02% v/v flanked by an EcoRI (59) and a SpeI(39) site which was inserted into Tween 40 (SOT). Bacto-agar (2% w/v) was added for solid media. Mat- EcoRI and SpeI-digested pKNSD44 resulting in pKNSD73. Finally, the ing, sporulation, and random spore analysis were performed as de- hybrid gene was inserted as a 0.9-kb BglII-NotI DNA fragment into the scribed by Gould et al. (1992). polylinker of pPIC3K (Invitrogen), resulting in pKNSD77. All of the Molecular Biological Techniques—Strain DH5a was used in all clon- GFP expression vectors were linearized by SalI digestion to target ing procedures involving plasmid propagation. Enzyme digests, cloning integration into the genomic PpHIS4 locus. techniques, plasmid isolations, polymerase chain reactions (PCRs) and Biochemical Assays, Sodium Dodecyl Sulfate-Polyacrylamide Gel Southern blotting were performed according to standard protocols. Electrophoresis (SDS-PAGE), and Western Blotting—Catalase and cy- DNA sequencing was performed according to Sanger et al. (1977), using tochrome c oxidase activities were determined according to Baudhuin et the Sequenase kit (U. S. Biochemical Corp.). al. (1964) and Madden and Storrie (1987), respectively. Protein concen- P. pastoris strains were transformed by electroporation according to trations were determined according to Smith et al. (1985) or Bradford Rickey (1990). DNA was isolated from yeast as described by Gould et al. (1976). (1992). SDS-PAGE and Western blotting were performed as described by Cloning and Sequencing of the PpPAS2 Gene—Strain PPY21 was Laemmli (1970) and Towbin et al. (1979), respectively. Blots were transformed with a P. pastoris genomic library described by Gould et al. incubated with primary antibodies, immune complexes were visualized (1992). The PpPAS2 gene was identified by functional complementation by the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium of the Pppas2 mutant, selecting for restoration of growth on SM and color reaction after incubation of blots with a goat anti-rabbit immuno- SOT media. Physical maps of the inserts from complementing plasmids globulin conjugated to alkaline phosphatase (dilution 1:5,000, Bio-Rad). were determined by restriction analysis. Fragments were subcloned Alternatively, immune complexes were detected by the ECL technique into plasmid pSG560 (Gould et al., 1992) and reintroduced into strain (Amersham Corp.) after incubation of blots with a protein A-horserad- PPY21. A 1.8-kb genomic DNA fragment with the ability to complement ish peroxidase conjugate (dilution 1:3,000, Bio-Rad). PPY21 for growth on methanol and oleate was cloned in both orienta- Antibodies—An AseI (filled in with Klenow polymerase)-HindIII tions into pBSII KS (Stratagene), yielding pBS-PAS2A and pBS- fragment of 1,717 base pairs (amino acids 8–455) of pBS-PAS2A was PAS2B. A set of plasmids with nested deletions generated by exonucle- cloned between the XbaI (filled in with Klenow polymerase) and Hin- ase III and S1 nuclease (Erase-a-Base kit, Promega, Madison, WI) was dIII sites of the pGEX-KG polylinker (Guan and Dixon, 1991). The used for sequencing both DNA strands. resulting glutathione S-transferase PpPas2p fusion (GST-PpPas2p) Construction of the Pppas2 Null Mutant—An EcoRI-AccI fragment of protein was synthesized in E. coli DH5a as described by Guan and 1,230 base pairs (nucleotides 116-1346), encompassing most of the Dixon (1991), except that no benzamidine was used, and the cells were PpPAS2 coding region, was replaced by a 2.0-kb EcoRI-HindIII frag- lysed by sonication. The largely insoluble GST-PpPas2p fusion protein ment containing the P. pastoris ARG4 gene (see Fig. 2). The AccI and was isolated by SDS-PAGE and cleaved by thrombin. The 52-kDa HindIII sites were filled in using Klenow polymerase. Strain PPY12 PpPas2p fragment was gel purified and used to immunize a rabbit was transformed with a linear DNA fragment containing the PpARG4 (Harlow and Lane, 1988). gene and PpPAS2 flanking regions. Transformants were selected for Anti-PpPer6p antibodies were a gift from Dr. J. M. Cregg (Oregon arginine prototrophy on SD plates and tested for growth on SM and Graduate Institute of Science and Technology, Portland, OR), and anti- SOT media. Cells that failed to utilize both carbon sources were ana- ScFox3p (anti-S. cerevisiae 3-ketoacyl-CoA thiolase) antibodies were a lyzed by diagnostic PCR and Southern blotting (Southern, 1975) to gift from Dr. W.-H. Kunau (Ruhr University, Bochum, Germany). Anti- confirm integration of the disruption construct at the correct chromo- somal locus. This strain was called SEW1 (Pppas2 null). PpPas8p, anti-PpAox1p (anti-P. pastoris alcohol oxidase), and anti-GFP Plasmids—Plasmid pJAH35-PAS2 was obtained by cloning the were generated as described by McCollum et al. (1993) and Monosov et 1.8-kb genomic DNA fragment containing the PpPAS2 gene as a SmaI al. (1996), respectively. PpPAS2, a Gene Involved in Peroxisomal Biogenesis 18975 FIG.1—continued sions in all peroxisomal subfractions were analyzed by SDS-PAGE and immunoblotting. Microscopy and Immunocytochemistry—GFP fluorescence in living cells was analyzed using the fluorescein isothiocyanate channel of a Photomicroscope III fluorescence microscope (Zeiss, Oberkochen, Ger- many) equipped with a Planopochromat 63/1.4 (oil) objective. Cells for electron microscopy were fixed with 1.5% w/v KMnO for 20 min at room temperature, washed extensively with water, resuspended in 0.5% w/v uranyl acetate, and incubated for 15 min at room temper- FIG.1. Nucleotide sequence of the PpPAS2 gene and the de- ature. Samples were embedded in Epon 812 after dehydration in a duced amino acid sequence. Shown is the translation of the PpPAS2 graded ethanol series. Ultrathin sections were examined in a Philips sequence from the ATG, at nucleotide 11, to the termination codon 300 electron microscope. (STOP), at nucleotides 1366–1368. The amino acids are in the one- Cells for immunocytochemistry were fixed in 3% v/v glutaraldehyde, letter code. Relevant restriction sites are indicated. 0.1 M sodium cacodylate, pH 7.2 for 30 min at 0 °C, dehydrated in a graded ethanol series, and embedded in Lowicryl K4 M. Immunolabel- Preparation of Crude Yeast Lysates—Yeast cells were harvested by ing was performed on ultrathin sections by the protein A-gold method centrifugation, resuspended in 3 volumes (compared with the volume of (Slot and Geuze, 1984), using anti-ScFox3p and anti-PpPas2p as pri- the pellet) of disruption buffer (20 mM Tris-Cl, pH 7.9, 10 mM MgCl ,1 mary antibodies and goat anti-rabbit immunoglobulin conjugated to mM EDTA, 5% v/v glycerol, 1 mM dithiothreitol, 0.3 M ammonium colloidal gold as secondary antibodies. Sections were poststained in sulfate, 0.2 mM phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin, and 5 0.5% w/v uranyl acetate and examined in a Philips 300 or CM10 elec- mg/ml aprotinin) and mixed with 4 volumes of acid-washed beads. Cell tron microscope. suspensions were vortexed for 1 min after which the tubes were placed on ice for 2 min. This treatment was repeated five times after which the RESULTS supernatants were removed. The beads were washed once with disrup- Cloning of PpPAS2 by Functional Complementation—Three tion buffer, and supernatants, representing the crude cell extracts, were Pppas2 mutants were isolated, selecting for cells deficient in pooled and stored at 220 °C. Subcellular Fractionations—Strains PPY4 (wild type for PpPAS2) peroxisome assembly (Gould et al., 1992), which were unable to and SEW1 (Pppas2 null mutant) were used for cell fractionation exper- grow on media containing oleic acid or methanol as sole carbon iments. Cells were cultured in 500 ml of YPD medium to near satura- sources. However, their growth was normal on glucose and tion, pelleted, resuspended in 2 liters of SM medium, and incubated for nonfermentable carbon sources such as glycerol, malate, etha- 24 h. Preparation of spheroplasts and cell homogenates, as well as the nol, and lactate. Through screening of a genomic DNA library, subsequent differential and density gradient centrifugation, were per- a 1.8-kb DNA fragment was obtained, which restored the abil- formed according to Monosov et al. (1996). Enriched organelle fractions (27,000 3 g pellet) of strains STW1 and ity of the mutant strain to utilize methanol and oleate. Se- SKF1, grown on SM medium, were diluted 10-fold either in 10 mM quencing of this DNA fragment revealed a 1,365-base pair open Tris-Cl, pH 8.5, or 1 M NaCl in 10 mM Tris-Cl, pH 8.5, or 0.1 M Na CO , 2 3 reading frame encoding a 455-amino acid protein (calculated pH 11.5, and incubated on ice for 1 h. Insoluble materials were pelleted molecular mass of 51,969 Da) with an estimated pI of 4.8 by centrifugation for1hat 100,000 3 g at 4 °C in a Beckman SW 50.1 (Fig. 1). rotor. Pellet fractions were resuspended in the appropriate buffer; after Deletion of PpPAS2 Abolishes Import of PTS1- and PTS2- centrifugation as above, the corresponding supernatants were pooled. Distributions of PpPas2p, PpPas8p, PpPer6p, catalase, and GFP fu- containing Reporter Proteins—The PpPAS2 gene was replaced 18976 PpPAS2, a Gene Involved in Peroxisomal Biogenesis FIG.2. Schematic representation of the PpPAS2 knockout construct. The gray bar represents the genomic fragment cloned by functional complementation of the Pppas2 mutant. The black bar marks the location of the PpPAS2 open reading frame. The PpPAS2 fragment that is replaced with the PpARG4 gene is indicated. Arrows indicate the direction of transcription. Ac, AccI; As, AseI; B, BamHI; E, EcoRI; H, HindII; HIII, HindIII; P, PstI; mcs, multiple cloning site of pBSII KS; bp, base pairs. FIG.4. Detection of GFP-PTS1 and PTS2-GFP in wild type and null mutant cells. The subcellular localization of the GFP-PTS1 and GFP-PTS2 reporter proteins was examined in living cells by fluores- cence microscopy. Wild type strain STW1 (panel A) and mutant strain FIG.3. Synthesis of PpPas2p in wild type and the null mutant. SEW2 (panel C), both expressing GFP-PTS1, were induced for 20 h in Crude extracts (100 mg of total protein) from wild type PPY1 cultured in methanol medium. Wild type strain STW2 (panel B) and mutant strain YPD (lane 1), YPM (lane 2), and the null mutant induced in YPM (lane SEW3 (panel D), both expressing PTS2-GFP, were induced for 20 h in 3) were subjected to SDS-PAGE. The proteins were transferred to oleate medium. Bar 5 1 mm. nitrocellulose and the blot incubated with anti-PpPas2p (dilution 1:5,000). Molecular mass markers are indicated on the right. The arrow marks PpPas2p, migrating at 52 kDa. as well as PTS2-GFP (Fig. 4D) were present in the cytoplasm as indicated by the diffuse fluorescent signal. with the PpARG4 gene (Fig. 2). The resulting Pppas2 null Peroxisomal Remnants Are Not Detectable in the Pppas2 Null mutant showed normal growth on glucose but was unable to Mutant—Ultrastructural examination of the null mutant did metabolize methanol and oleate, as observed for the original not reveal any morphologically distinguishable peroxisomes on mutants. In a genetic cross between the PpPas2 null mutant methanol (Fig. 5C) or oleate (results not shown). In wild type and the PpPas2 mutant, no complementation was observed for cells, the peroxisomal compartment was clearly visible and had growth on methanol or oleate. This indicates that the identified a characteristic morphology when grown in methanol (Fig. 5A) open reading frame was not that of a suppressor gene. Rein- and oleic acid (Fig. 5B). Large, clustered peroxisomes filled troduction of PpPAS2 into the null mutant restored the ability with alcohol oxidase were seen in methanol-grown cells; and to grow on methanol and oleate (results not shown). Notably, small, more abundant peroxisomes, identified as such by the the cloned 59-noncoding region of only 90 nucleotides was suf- presence of 3-ketoacyl-CoA thiolase, were evident in oleate- ficient to direct expression of the PpPAS2 gene on both oleate grown cells. and methanol (see below). An enriched organelle fraction was prepared from wild type Antibodies raised against PpPas2p specifically detected a cells induced on methanol by differential centrifugation and protein of approximately 52 kDa in lysates of wild type P. subjected to isopycnic centrifugation on a continous Nycodenz pastoris cells grown on YPD (Fig. 3, lane 1), and only a mod- gradient. Peroxisomes migrated to their buoyant density of erate 3–5-fold induction of the PpPas2p was noted on methanol 1.23 g/ml (fractions 2–6) as judged by the distribution of cata- (Fig. 3, lane 2) and oleate (results not shown). As expected, no lase (Fig. 6A), well separated from the bulk of mitochondria PpPas2p was detected in lysates from the null mutant (Fig. 3, (fractions 12–20) identified by cytochrome c oxidase. Or- lane 3). ganelles from the null mutant, fractionated in the same way, The import of peroxisomal matrix proteins was assessed by lacked normal peroxisomes, whereas mitochondria were found determining the intracellular location of GFP fused either to a at their normal position in the gradient (Fig. 6C). Only a small PTS1 or PTS2 sequence. A GFP-PTS1 fusion protein was ex- amount of catalase activity was recovered in organellar frac- pressed in strains STW1 (producing PpPas2p and GFP-PTS1) tions of lower density in the Nycodenz gradient. and SEW2 (producing GFP-PTS1 but lacking PpPas2p). Upon Equal portions of the gradient fractions from both wild type induction on methanol, the GFP-PTS1 protein was directed to and null mutant cells were analyzed by immunoblotting. Alco- the peroxisomes of STW1 cells as judged by the intense fluo- hol oxidase, PpPas8p, and PpPas2p were predominantly per- rescent spots representing the large clustered peroxisomes oxisomal (Fig. 6B), with some trailing of PpPas2p and PpPas8p (Fig. 4A). These results are in agreement with data published into the lighter parts of the gradient, a phenomenon observed by Monosov et al. (1996). Likewise, a hybrid protein consisting frequently with membrane-associated proteins. In the gradient of the NH -terminal 16 amino acids of S. cerevisiae thiolase and from the null mutant, none of these proteins were detected in GFP (PTS2-GFP) was targeted to the peroxisomes in oleate- the denser parts of the gradient where peroxisomes usually grown STW2 (expressing PpPas2p and PTS2-GFP) (Fig. 4B). band (Fig. 6, C and D). However, as observed in the wild type, This implies that the sequence identified as the PTS for thio- traces of PpPas8p were found in gradient fractions of lower lase is also properly recognized as such in P. pastoris. In strains density in the null mutant (Fig. 6, B and D). SEW2 and SEW3, lacking PpPas2p, both GFP-PTS1 (Fig. 4C) PpPas2p Is a Peroxisomal Membrane Protein—The location PpPAS2, a Gene Involved in Peroxisomal Biogenesis 18977 FIG.5. Ultrastructural analysis of wild type and null mutant cells. Sec- tions of KMnO -fixed, methanol-grown cells of wild type PPY1 (panel A) and the null mutant (panel C) are shown. Panel B, immunocytochemical detection of the b-oxidation enzyme 3-ketoacyl-CoA thio- lase using anti-ScFox3p in a section of p-formaldehyde-fixed, oleic acid-grown cells of PPY1. Note that the morphologi- cal appearance of the peroxisomal com- partment differs, with a few large clus- tered peroxisomes in methanol-induced cells (panel A) and smaller, more abun- dant, and dispersed peroxisomes in oleate-induced cells (panel B). No peroxi- somal structures are visible in the null mutant (panel C). The vesicles adjacent to the nuclear membrane are also observed routinely in wild type P. pastoris cells (Gould et al., 1992) and are not thought to be of peroxisomal origin. Panels D and E, immunocytochemical detection of PpPas2p in methanol-grown cells using anti-PpPas2p or the appropriate preim- mune serum as the control (panel F). N, nucleus; V, vacuole; M, mitochondrion. Bar, 0.5 mm. of PpPas2p within the peroxisomes was analyzed by immuno- erly targeted to peroxisomes. cytochemistry and organelle subfractionation. Sections of To address the subperoxisomal localization of the 1–40-GFP methanol-grown wild type cells were incubated with anti- fusion, crude organellar fractions were prepared from strains PpPas2p followed by gold-conjugated protein A. The gold par- STW1 and SKF1 grown on SM medium, and the extractability ticles almost exclusively decorated the peroxisomal membrane of the fusion proteins after low salt, high salt, or carbonate (Fig. 5, D and E). A control incubation using the preimmune treatment was analyzed by Western blotting. In strain STW1, antiserum showed no labeling of the peroxisomal membrane the GFP-PTS1 fusion protein was completely soluble after car- (Fig. 5F). bonate treatment, and even after low and high salt extraction Strain STW1 was grown in SM medium, and crude organelle the bulk of the fusion protein was found in the supernatants as pellets were fractionated into soluble and insoluble fractions expected for soluble matrix proteins (Fig. 7A). In strain SKF1, after treatment with 10 mM Tris, pH 8.5 (low salt), 1 M NaCl in however, the 1–40-GFP fusion protein was insoluble under all three conditions (Fig. 7B), behaving like the full-length 10 mM Tris, pH 8.5 (high salt), or 0.1 M Na CO as described 2 3 under “Materials and Methods.” PpPas2p, PpPas8p, and PpPas2p, as well as PpPas8p and PpPer6p (Fig. 7A). PpPer6p were insoluble under all three conditions (Fig. 7A), PpPas2p Is Homologous to S. cerevisiae Pas3p Protein—A thus behaving like integral membrane proteins. Catalase, a data base search using the BLAST program revealed that the marker for soluble matrix proteins, was found in the superna- PpPas2p protein displays a high similarity to ScPas3p, a per- tant under all conditions tested. oxisomal integral membrane protein (Ho¨hfeld et al., 1991) but The NH -terminal 40 Amino Acids of PpPas2p Contain a not to other proteins. From the sequence alignment (Fig. 9A)an Peroxisomal Targeting Signal—Analysis of the PpPas2p amino overall identity of 35% was determined. There is a significant acid sequence did not reveal any known PTS. Several NH - similarity (62%) over the entire length of PpPas2p and terminal fragments of PpPAS2p, varying in length from 40 to ScPas3p, but a number of segments that are highly conserved 110 amino acids, were tested for their ability to target the can be distinguished (e.g. PpPas2p amino acid residues 49–64, reporter protein GFP to peroxisomes. As shown in Fig. 8, the 70–78, 215–233, and 442–453). The putative membrane-span- smallest hybrid protein, consisting of the NH -terminal 40 ning domain (residues 18–39) and membrane-anchoring do- amino acids of PpPas2p fused to GFP (1–40-GFP), was prop- main (residues 135–153) of ScPas3p do not seem to be partic- 18978 PpPAS2, a Gene Involved in Peroxisomal Biogenesis FIG.7. Subperoxisomal localization of PpPas2p and heterolo- gous proteins in strains STW1 (GFP-PTS1) and SKF1 (1–40- GFP). Crude organelle fractions were prepared from methanol-induced strains STW1 (panel A) and SKF1 (panel B) and subfractionated into an insoluble pellet fraction (P) and a soluble fraction (S) after treatment with NaCl, Tris, or Na CO (Carb.) as described under “Materials and 2 3 Methods.” Distributions of PpPas2p, PpPas8p, PpPer6p, catalase, and the GFP fusion proteins between supernatant and membranous pellet fractions were examined by immunoblotting. The distribution of the four endogenous proteins was identical for both strains and is shown for strain STW1. Antibodies used were a-PpPas2p (dilution 1:5,000), a-PpPas8p (dilution 1:10,000), a-PpPer6p (dilution 1:2,000), a-catalase (dilution 1:5,000), and a-GFP (dilution 1:5,000). FIG.6. Nycodenz density gradient fractionation of organelles from wild type and null mutant cells. Crude organelle fractions FIG.8. A PTS resides near the NH terminus of PpPAS2p. from methanol-induced cells, consisting primarily of peroxisomes and Localization of a hybrid protein consisting of the NH -terminal 40 mitochondria, were fractionated on Nycodenz gradients as described amino acids of PpPAS2p and the GFP (1–40-GFP) was determined by under “Materials and Methods.” The distribution of peroxisomal cata- confocal microscopy. P. pastoris cells producing the hybrid protein were lase and mitochondrial cytochrome c oxidase in gradients of wild type grown in synthetic media containing methanol as sole carbon source. strain PPY4 (panel A) and null mutant SEW1 (panel C) is shown. Note that the cells have large, clustered peroxisomes (panel A) which Enzyme units are expressed as relative specific activities, reflecting the colocalize with the GFP fluorescence (panel B). percentage enzyme activity in a given fraction relative to the total amount of protein contained in the gradient. Equal volumes of fractions 1995; Liu et al., 1995) reminiscent of the structures seen in from the gradients of wild type PPY4 (panel B) and the null mutant SEW1 (panel D) were analyzed by immunoblotting. Blots were incu- human patients suffering from generalized peroxisomal disor- bated with antisera against PpPas8p (a-PpPas8p, dilution 1:5,000), ders. The Pppas2 null mutant described in this study is an PpPas2p (a-PpPas2p, dilution 1:5,000), and against alcohol oxidase exception to this rule since it lacks morphologically detectable (a-PpAox1p, dilution 1:20,000). No PpAox1p could be detected in gra- peroxisomes (Fig. 5C) even upon induction on methanol or dient fractions of the null mutant. oleate, both of which are known to induce peroxisomes. In cell fractionation experiments normal peroxisomes could not be ularly conserved in PpPas2p. The hydrophobicity plot, detected either (Fig. 6, C and D). Traces of the peroxisomal prepared according to Kyte and Doolittle (1982), shows that the membrane-associated protein PpPas8p, however, were found NH -terminal portion of PpPas2p is not as hydrophobic as the in fractions of lower density near the top of the gradient in both corresponding part of ScPas3p (Fig. 9B, compare with Fig. 5. in wild type (Fig. 6B) and the null mutant (Fig. 6D). Whether this Ho¨hfeld et al. (1991)). However, two hydrophobic domains seem represents a real peroxisomal (precursor) fraction or nonspe- to be formed by amino acid residues 66–79 and 149–174. cific binding of PpPas8p to mitochondria or other membranous DISCUSSION particles is unknown. In the Pppas2 null mutant, marker pro- Most of the P. pastoris pas and per mutants characterized to teins containing a PTS1 or a PTS2 sequence were found to be date harbor morphologically and biochemically detectable per- localized to the cytoplasm (Fig. 4). These data suggest that oxisome ghosts or membrane remnants (McCollum et al., 1993; PpPas2p is involved in the early stages of peroxisome biogen- Spong and Subramani, 1993; Heyman et al., 1994; Kalish et al., esis. Therefore, we propose that the proper functioning of PpPAS2, a Gene Involved in Peroxisomal Biogenesis 18979 PpPas2p is a prerequisite for the assembly of matrix, and at least some membrane, proteins into peroxisomes. PpPas2p is homologous to Pas3p of S. cerevisiae (ScPas3p), which has been shown by Ho¨hfeld et al. (1991) to be a peroxi- somal integral membrane protein. Immunocytochemical detec- tion of PpPas2p revealed that it is also associated with the peroxisomal membrane (Fig. 5, D and E). This was further substantiated by biochemical experiments that showed that the protein behaves like an integral membrane protein during subfractionation of an organelle pellet fraction (Fig. 7). Earlier experiments show that there are at least two differ- ent signals that target proteins to the peroxisomal matrix. PMPs must use a different targeting signal, which remains to be defined. Proteins that reside in the peroxisomal membrane can be envisaged as having two components: an mPTS, which targets the protein to the peroxisome, and a transmembrane domain (TMD), which anchors the protein in the membrane, or a protein-protein interaction domain that anchors it to the membrane through other peroxisomal integral membrane pro- teins. These two components could either be separate or overlapping. The PTS in ScPas3p has been described to lie in the NH - terminal half of the protein, a region that encompasses a pu- tative transmembrane domain (Ho¨hfeld et al., 1992). Our result on the targeting of the 1–40-GFP fusion reveals the presence of an mPTS in the NH -terminal 40 amino acids of PpPas2p (Fig. 8). In contrast to ScPas3p, the first 40 amino acids of PpPas2p do not contain any obvious TMD (Figs. 1 and 9B). Yet the 1–40-GFP fusion is directed faithfully to the peroxisomal mem- brane, where it behaves exactly like the full-length PpPas2p in terms of its inextractability with sodium carbonate (Fig. 7). This suggests that these 40 amino acids have both an mPTS as well as a domain that allows the fusion to interact tightly with the peroxisomal membrane or with one or more PMPs. The sequence and experimental data seem contradictory, having a protein behaving like an integral membrane protein without predicted transmembrane segments. On one hand, several (peroxisomal) proteins have been shown to be sodium carbon- ate-inextractable (Tan et al., 1995; Erdmann and Blobel, 1995) from the membranes, whereas no clear transmembrane seg- ments could be detected by sequence analysis. On the other hand, some proteins with strong protein-protein interactions might resist the sodium carbonate extraction procedure and therefore are falsely classified as integral membrane proteins. Based on the relatively low content of hydrophobic residues and the presence of a highly positively charged domain in the FIG.9. Sequence comparison and hydrophobicity analysis of NH -terminal 40 amino acids, we favor the possibility of a PpPas2p and ScPas3p. The amino acid sequences were aligned using strong interaction between this sequence in PpPas2p and an- the BESTFIT program (panel A). Identical residues are indicated by an other peroxisomal integral membrane protein. asterisk below the sequence; a dot represents aligned residues that are Based on the proposed topology of ScPAS3p, we expected similar. Two hydrophobic domains, identified by Ho¨hfeld et al. (1991), that the GFP segment in the 1–40-GFP hybrid protein would in ScPas3p are underlined. Similarity rules: G 5 A 5 S; V 5 I 5 L 5 M 5 F 5 Y 5 W; K 5 r 5 H; D 5 E 5 Q 5 N; S 5 T 5 Q 5 N. Panel face the cytosol. Therefore, we performed a proteinase K pro- B, hydrophobicity plot of PpPas2p and ScPas3p according to Kyte and tection experiment on a crude organellar fraction to determine Doolittle (1982), using a window size of 19 amino acids. Two hydropho- whether it would be sensitive to proteolytic degradation. The bic domains are revealed in PpPas2p, a 13-amino acid stretch at posi- tion 66–79, and a potential membrane-spanning region at position hybrid protein appeared to be resistant to proteolytic degrada- 149–174. tion, whether or not the organelles were disrupted by Triton X-100 prior to the proteinase K treatment. This suggests that GFP itself is highly resistant to proteolytic degradation, and 1994). In a more detailed analysis of the targeting information therefore the results were not conclusive. in CbPMP47 it was shown that a stretch of 20 amino acids in A putative mPTS has been identified in a Candida boidinii the intervening loop between transmembrane domains 4 and 5 protein, CbPMP47. This protein is homologous to a family of was able to direct a soluble reporter protein to the peroxisomal mitochondrial solute transporters that span the membrane six membrane of S. cerevisiae (McNew and Goodman, 1996). It is times (Kuan and Saier, 1993). The targeting information on interesting to note that in both PpPas2p and in CbPMP47 the this protein was localized to a region of the protein containing mPTS does not include any predicted TMD. This supports the transmembrane domains 4 and 5 and an intervening 20-amino idea proposed above that the mPTS and TMD are separable acid loop facing the peroxisomal matrix (McCammon et al., entities and that it is the mPTS that directs a TMD in a protein 18980 PpPAS2, a Gene Involved in Peroxisomal Biogenesis Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–267 to the peroxisomal membrane. This model predicts that the Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, pp. 92–114, TMDs of PMPs would have no higher affinity for the peroxiso- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY mal membrane relative to other subcellular membranes. Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Nature 373, 663–664 Heyman, J. A., Monosov, E., and Subramani, S. (1994) J. Cell Biol. 127, 1259–1273 What are the features common to mPTSs from different Ho¨hfeld, J., Veenhuis, M., and Kunau, W. H. (1991) J. Cell Biol. 114, 1167–1178 proteins? A comparison of the 20-amino acid segment of Cb- Ho¨hfeld, J., Mertens, D., Wiebel, F. F., and Kunau, W.-H. (1992) in Membrane Biogenesis and Protein Targeting (Neupert, W., and Lill, R., eds) pp. 185–207, PMP47 and the NH termini of PpPas2p and ScPas3p reveals Elsevier, New York a block of five amino acids (of which four are positively charged) Imanaka, T., Shiina, Y., Takano, T., Hashimoto, T., and Osumi, T. (1996) J. Biol. in CbPMP47 which is also observed in both PpPas2p and Chem. 271, 3706–3713 Kalish, J. E., Theda, C., Morrell, J. C., Berg, J. M., and Gould, S. J. (1995) Mol. ScPas3p (QIKRR) at positions 53–57 and 59–63, respectively, Cell. Biol. 15, 6406–6419 being 100% conserved between these Pas proteins. Notably, Keller, G. A., Krisans, S. K., Gould, S. J., Sommer, J. M., Wang, C. C., Schliebs, W., this sequence is excluded from the first 40 amino acids of Kunau, W.-H., Brody, S., and Subramani, S. (1991) J. Cell Biol. 114, 893–904 Kuan, J., and Saier, M. J. (1993) Res. Microbiol. 144, 671–672 PpPas2p, which are able to direct GFP to the peroxisomal Kunau, W.-H, Kionka, C., Ledebur, A., Mateblowski, M., Moreno de la Garza, M., membrane. Another positively charged sequence (RRNKKK) Schultz-Borchard, U., Thieringer, R., and Veenhuis, M. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 128–140, Springer present at position 10–15 might be involved in targeting of Verlag, Berlin PpPas2p, although this sequence is not particularly conserved Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105–132 in ScPas3p. Laemmli, U. K. (1970) Nature 227, 680–685 Lazarow, P. B., and Moser, H. W. (1989) in The Metabolic Bases of Inherited Mutational analysis of the first 40 amino acids of PpPas2p Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 6th Edition, will reveal whether the mPTS and peroxisomal membrane pp. 1479–1509, McGraw Hill, New York Liu, H., Tan, X., Veenhuis, M., McCollum, D., and Cregg, J. M. (1992) J. Bacteriol. interaction domain are overlapping or physically separable 174, 4943–4951 entities. It may also help address whether an mPTS and per- Liu, H., Tan, X., Russell, K. A., Veenhuis, M., and Cregg, J. M. (1995) J. Biol. oxisomal membrane interaction domain need to coexist for Chem. 270, 10940–10951 Madden, E. A., and Storrie, B. (1987) Anal. Biochem. 163, 350–357 proper targeting to the peroxisomal membrane. Marzioch, M., Erdmann, R., Veenhuis, M., and Kunau, W.-H. (1994) EMBO J. 13, Studies on the import of PMPs into peroxisomes in vitro have 4908–4917 been undertaken without any knowledge of the mPTSs in- McCammon, M. T., McNew, J. A., Willy, P. J., and Goodman, J. M. (1994) J. Cell Biol. 124, 915–925 volved in the targeting process (Diestelkotter and Just, 1993; McCollum, D., Monosov, E., and Subramani, S. (1993) J. Cell Biol. 121, 761–774 Imanaka et al., 1996). These experiments reveal that import of McNew, J. A., and Goodman, J. M. (1996) Trends Biochem. Sci. 21, 54–58 Monosov, E. Z., Wenzel, T. J., Lu¨ers, G. H., Heyman, J. A., and Subramani, S. such proteins is dependent on time and temperature, does not (1996) J. Histochem. Cytochem. 44, 581–589 require ATP or GTP, and is not inhibited by N-ethylmaleimide Motley, A., Hettema, E., Distel, B., and Tabak, H. F. (1994) J. Cell Biol. 125, treatment of either the soluble components or of the isolated 755–767 Nuttley, W. M., Brade, A. M., Gaillardin, C., Eitzen, G. A., Glover, J. R., peroxisomes. In contrast, the import of the peroxisomal matrix Aitchinson, J. D., and Rachubinski, R. A. (1993) Yeast 9, 507–517 marker, firefly luciferase, into peroxisomes of permeabilized Osumi, T., Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikta, M., cells is dependent on ATP hydrolysis and is blocked by N- Miyazawa, S., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 181, 947–954 ethylmaleimide pretreatment of the cytosol-depleted cells Rapp, S., Soto, U., and Just, W. W. (1993) Exp. Cell Res. 205, 59–65 (Rapp et al., 1993; Wendland and Subramani, 1993). These Rickey, C. (1990) Mol. Biol. Rep. 9, 2–3 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, properties, as well as the import of PMPs into peroxisome 5463–5467 ghosts in yeast and human cell lines deficient in the import of Slawecki, M. L., Dodt, G., Steinberg, S., Moser, A. B., Moser, H. W., and Gould, S. PTS1- and PTS2-containing proteins, imply that the mecha- J. (1995) J. Cell Sci. 108, 1817–1829 Slot, J. W., and Geuze, H. J. 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Isolation and Characterization of Pas2p, a Peroxisomal Membrane Protein Essential for Peroxisome Biogenesis in the Methylotrophic Yeast Pichia pastoris

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 31, Issue of August 2, pp. 18973–18980, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Isolation and Characterization of Pas2p, a Peroxisomal Membrane Protein Essential for Peroxisome Biogenesis in the Methylotrophic Yeast Pichia pastoris* (Received for publication, April 5, 1996) Erik A. C. Wiemer‡§, Georg H. Lu¨ ers‡, Klaas Nico Faber‡, Thibaut Wenzel‡, Marten Veenhuis , and Suresh Subramani‡ From the ‡Department of Biology, University of California at San Diego, La Jolla, California 92093-0322, and the ¶Laboratory for Electron Microscopy, University of Groningen, Biological Center, 9751 NN Haren, The Netherlands The pas2 mutant of the methylotrophic yeast Pichia involvement in H O metabolism and the b-oxidation of fatty 2 2 pastoris is characterized by a deficiency in peroxisome acids (Van den Bosch et al., 1992; Wiemer and Subramani, biogenesis. We have cloned the PpPAS2 gene by func- 1994). The importance of peroxisomes is emphasized by the tional complementation and show that it encodes a pro- existence of severely debilitating, and often lethal, human dis- tein of 455 amino acids with a molecular mass of 52 kDa. eases in which peroxisome biogenesis is impaired (Lazarow and In a Pppas2 null mutant, import of both peroxisomal Moser, 1989). In fungi, peroxisomes are the sole site for b-oxi- targeting signal 1 (PTS1)- and PTS2-containing proteins dation of fatty acids and, in methylotrophic yeasts, are essen- is impaired as shown by biochemical fractionation and tial for the oxidation of methanol (Kunau et al., 1987; Veenhuis, fluorescence microscopy. No morphologically distin- 1992). Much progress has been made in recent years delineat- guishable peroxisomal structures could be detected by ing the molecular requirements for import of peroxisomal ma- electron microscopy in Pppas2 null cells induced on trix proteins (metabolic enzymes). Typically, these proteins are methanol and oleate, suggesting that PpPas2p is in- synthesized on cytosolic polysomes and imported posttransla- volved in the early stages of peroxisome biogenesis. tionally without further modifications. Two evolutionary con- PpPas2p is a peroxisomal membrane protein (PMP) and served peroxisomal targeting signals (PTSs) for matrix pro- is resistant to extraction by 1 M NaCl or alkaline sodium teins have been identified (Subramani, 1993). Most frequently carbonate, suggesting that it is a peroxisomal integral encountered is PTS1, consisting of a COOH-terminal tripeptide membrane protein. Two hydrophobic domains can be with the consensus sequence S/A/C-K/R/H/-L/M (Gould et al., distinguished which may be involved in anchoring 1989; Keller et al., 1991). PTS2 is found at the amino termini of PpPas2p to the peroxisomal membrane. PpPas2p is ho- a smaller subset of peroxisomal proteins and conforms to the mologous to the Saccharomyces cerevisiae Pas3p. The consensus RL-X -H/QL (Swinkels et al., 1991; Osumi et al., first 40 amino acids of PpPas2p, devoid of the hydropho- 1991; Erdmann, 1994; Glover et al., 1994; Faber et al., 1995). bic domains, are sufficient to target a soluble fluores- cent reporter protein to the peroxisomal membrane, Separate import pathways exist for PTS1- and PTS2-contain- with which it associates tightly. A comparison with the ing proteins since both in yeast and mammalian cells import membrane peroxisomal targeting signal of PMP47 of defects have been observed excluding either PTS1- or PTS2- Candida boidinii revealed a stretch of positively containing proteins from the peroxisome (McCollum et al., charged amino acids common to both sequences. The 1993; Motley et al., 1994; Marzioch et al., 1994; Slawecki et al., role of peroxisomal membrane targeting signals and 1995; Zhang and Lazarow, 1995). Functional complementation transmembrane domains in anchoring PMPs to the per- of the yeasts has revealed the PTS1 (PpPas8p, ScPas10p, oxisomal membrane is discussed. HpPer3p, and YlPay32p) and PTS2 receptors (ScPas7p or ScPeb1p) that specifically interact with their cognate signals (McCollum et al., 1993; Van der Leij et al., 1993; Marzioch et Peroxisomes (microbodies) comprise an inducible and versa- al., 1994; Zhang and Lazarow, 1995, 1996; Terlecky et al., tile eukaryotic subcellular compartment that plays a key role 1995). in a number of metabolic pathways. A general feature is its Besides the yeast mutants with a selective import defect for matrix proteins, a large collection of strains has been described which show a general defect in peroxisome assembly (pas, per * This work was supported in part by a grant from the Human pay,or peb mutants; Erdmann et al., 1989; Cregg et al., 1990; Frontier Science Program Organization (HFSPO) and the American Gould et al., 1992; Liu et al., 1992; Van der Leij et al., 1992; Heart Association (to E. A. C. W.), an HFSPO fellowship (to K. N. F.), a fellowship from the Deutsche Forschungsgemeinshaft (to G. L.), a fel- Elgersma et al., 1993; Nuttley et al., 1993; Zhang and Lazarow, lowship from the European Molecular Biology Organization (to T. W.), 1993). In these strains the bulk of the peroxisomal matrix and by National Institutes of Health Grant NIHDK41737 and National proteins is found in the cytosol. Some contain peroxisomes with Science Foundation Grant MCB9316018 (to S. S.). The costs of publi- an aberrant morphology reminiscent of the peroxisomal ghost cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” structures found in fibroblast cell lines derived from patients in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with peroxisomal disorders (Wiemer et al., 1989). The nucleotide sequence(s) reported in this paper has been submitted TM to the GenBank /EBI Data Bank with accession number(s) Z72390. § Present address: Institute for Hematology, Erasmus University, Rotterdam, The Netherlands. The abbreviations used are: PTS(s), peroxisomal targeting signal(s); To whom correspondence should be addressed: Dept. of Biology, PMP, peroxisomal membrane protein; mPTS, peroxisomal membrane UCSD, Rm. 4314, Bonner Hall, 9500 Gilman Dr., La Jolla, CA 92093- targeting signal; PCR, polymerase chain reaction; kb, kilobase; GFP, 0322. Tel.: 619-534-2327; Fax: 619-534-0053; E-mail: subra@ green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; jeeves.ucsd.edu. TMD, transmembrane domain; PAS, peroxisome assembly. This is an open access article under the CC BY license. 18973 18974 PpPAS2, a Gene Involved in Peroxisomal Biogenesis TABLE I P. pastoris strains Strain Genotype (expression construct) Source PPY1 21-1 Wild type Y-11430, NRRL PPY4 Pphis4 Gould et al. (1992) PPY12 Pphis4, Pparg4 Gould et al. (1992) PPY21 Pppas2, Pparg4 Gould et al. (1992) SEW1 (Pppas2 null) Pphis4, Pppas2<PpARG4 This paper SEW2 Pppas2<PpARG4, Pphis4::pTW51 (P GFP-PTS1) This paper AOXI SEW3 Pppas2<PpARG4, Pphis4<pTW66 (P PTS2-GFP) This paper GAPDH STW1 Pparg4, Pphis4<pTW51 (P GFP-PTS1) This paper AOXI STW2 Pparg4, Pphis4<pTW66 (P PTS2-GFP) This paper GAPDH SKF1 Pparg4, Pphis4<pKNSD77 (P 1-40-GFP) This paper AOXI fragment (polylinker sites) into the EcoRI site (blunted with Klenow Complementing genes have been isolated and characterized polymerase) of pJAH35, a pBR322-based vector containing the PpHIS4 for most of these mutants. Several of the encoded proteins were gene and a P. pastoris autonomously replicating sequence (PARS2, shown to be membrane-associated. The functions and molecu- Cregg et al., 1985). lar requirements for targeting of these peroxisomal membrane Plasmid pTW51, containing the green fluorescent protein-PTS1 proteins (PMPs), which do not contain a PTS1 or PTS2, are (GFP-PTS1) construct under control of the methanol-inducible alcohol largely unknown. oxidase promoter (P ), was obtained by cloning an EcoRI PCR prod- AOX1 Here we report the cloning of the Pichia pastoris PAS2 gene, uct of the GFPS65T mutant allele (Heim et al., 1995) in the P. pastoris integrating vector pHIL-D2 (Invitrogen, San Diego). The primer at the encoding a peroxisomal membrane protein (PpPas2p) which is 39 end contained the appropriate codons to include SKL (PTS1 signal) in essential for peroxisome biogenesis. The phenotype of the Pp- the predicted translation product (Monosov et al., 1996). Plasmid pas2 null strain is examined, and the peroxisomal membrane- pTW66, containing the PTS2-GFP construct under control of the con- targeting signal (mPTS) of PpPas2p is delineated. stitutive glyceraldehyde 3-phosphate dehydrogenase promoter (P ; GAPDH gift of Dr. J. M. Cregg, Oregon Graduate Institute of Science and MATERIALS AND METHODS Technology, Portland, OR) was constructed by cloning the GFPS65T Yeast Strains and Culture Conditions—The P. pastoris strains used allele in-frame with a part of the ScFOX3 sequence (Glover et al., 1994) in this study are listed in Table I. Yeast strains were grown at 30 °C in encoding the NH -terminal 16 amino acids of the Saccharomyces cer- YPD (1% w/v yeast extract, 2% w/v Bacto-peptone, 2% w/v dextrose), evisiae 3-oxoacyl-CoA thiolase in vector pHIL-D2. YPM (1% w/v yeast extract, 2% w/v Bacto-peptone, 0.5% v/v methanol), A hybrid gene encoding the NH -terminal 40 amino acids of PpPas2p YPOT (1% w/v yeast extract, 2% w/v Bacto-peptone, 0.2% v/v oleate, and GFP (1–40-GFP) was constructed as follows. By PCR, an Asp-718 0.02% v/v Tween 40), or in synthetic medium consisting of 0.67% w/v and a BglII site were introduced upstream of the PpPAS2 open reading yeast nitrogen base, supplemented with 50 mg/ml of the appropriate frame present in pBSIIKS, resulting in pKNSD44. A second PCR was amino acids and with one of the following carbon sources: 2% w/v performed to amplify a DNA fragment encoding the GFPS65T allele dextrose (SD), 0.5% v/v methanol (SM), or 0.2% v/v oleate and 0.02% v/v flanked by an EcoRI (59) and a SpeI(39) site which was inserted into Tween 40 (SOT). Bacto-agar (2% w/v) was added for solid media. Mat- EcoRI and SpeI-digested pKNSD44 resulting in pKNSD73. Finally, the ing, sporulation, and random spore analysis were performed as de- hybrid gene was inserted as a 0.9-kb BglII-NotI DNA fragment into the scribed by Gould et al. (1992). polylinker of pPIC3K (Invitrogen), resulting in pKNSD77. All of the Molecular Biological Techniques—Strain DH5a was used in all clon- GFP expression vectors were linearized by SalI digestion to target ing procedures involving plasmid propagation. Enzyme digests, cloning integration into the genomic PpHIS4 locus. techniques, plasmid isolations, polymerase chain reactions (PCRs) and Biochemical Assays, Sodium Dodecyl Sulfate-Polyacrylamide Gel Southern blotting were performed according to standard protocols. Electrophoresis (SDS-PAGE), and Western Blotting—Catalase and cy- DNA sequencing was performed according to Sanger et al. (1977), using tochrome c oxidase activities were determined according to Baudhuin et the Sequenase kit (U. S. Biochemical Corp.). al. (1964) and Madden and Storrie (1987), respectively. Protein concen- P. pastoris strains were transformed by electroporation according to trations were determined according to Smith et al. (1985) or Bradford Rickey (1990). DNA was isolated from yeast as described by Gould et al. (1976). (1992). SDS-PAGE and Western blotting were performed as described by Cloning and Sequencing of the PpPAS2 Gene—Strain PPY21 was Laemmli (1970) and Towbin et al. (1979), respectively. Blots were transformed with a P. pastoris genomic library described by Gould et al. incubated with primary antibodies, immune complexes were visualized (1992). The PpPAS2 gene was identified by functional complementation by the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium of the Pppas2 mutant, selecting for restoration of growth on SM and color reaction after incubation of blots with a goat anti-rabbit immuno- SOT media. Physical maps of the inserts from complementing plasmids globulin conjugated to alkaline phosphatase (dilution 1:5,000, Bio-Rad). were determined by restriction analysis. Fragments were subcloned Alternatively, immune complexes were detected by the ECL technique into plasmid pSG560 (Gould et al., 1992) and reintroduced into strain (Amersham Corp.) after incubation of blots with a protein A-horserad- PPY21. A 1.8-kb genomic DNA fragment with the ability to complement ish peroxidase conjugate (dilution 1:3,000, Bio-Rad). PPY21 for growth on methanol and oleate was cloned in both orienta- Antibodies—An AseI (filled in with Klenow polymerase)-HindIII tions into pBSII KS (Stratagene), yielding pBS-PAS2A and pBS- fragment of 1,717 base pairs (amino acids 8–455) of pBS-PAS2A was PAS2B. A set of plasmids with nested deletions generated by exonucle- cloned between the XbaI (filled in with Klenow polymerase) and Hin- ase III and S1 nuclease (Erase-a-Base kit, Promega, Madison, WI) was dIII sites of the pGEX-KG polylinker (Guan and Dixon, 1991). The used for sequencing both DNA strands. resulting glutathione S-transferase PpPas2p fusion (GST-PpPas2p) Construction of the Pppas2 Null Mutant—An EcoRI-AccI fragment of protein was synthesized in E. coli DH5a as described by Guan and 1,230 base pairs (nucleotides 116-1346), encompassing most of the Dixon (1991), except that no benzamidine was used, and the cells were PpPAS2 coding region, was replaced by a 2.0-kb EcoRI-HindIII frag- lysed by sonication. The largely insoluble GST-PpPas2p fusion protein ment containing the P. pastoris ARG4 gene (see Fig. 2). The AccI and was isolated by SDS-PAGE and cleaved by thrombin. The 52-kDa HindIII sites were filled in using Klenow polymerase. Strain PPY12 PpPas2p fragment was gel purified and used to immunize a rabbit was transformed with a linear DNA fragment containing the PpARG4 (Harlow and Lane, 1988). gene and PpPAS2 flanking regions. Transformants were selected for Anti-PpPer6p antibodies were a gift from Dr. J. M. Cregg (Oregon arginine prototrophy on SD plates and tested for growth on SM and Graduate Institute of Science and Technology, Portland, OR), and anti- SOT media. Cells that failed to utilize both carbon sources were ana- ScFox3p (anti-S. cerevisiae 3-ketoacyl-CoA thiolase) antibodies were a lyzed by diagnostic PCR and Southern blotting (Southern, 1975) to gift from Dr. W.-H. Kunau (Ruhr University, Bochum, Germany). Anti- confirm integration of the disruption construct at the correct chromo- somal locus. This strain was called SEW1 (Pppas2 null). PpPas8p, anti-PpAox1p (anti-P. pastoris alcohol oxidase), and anti-GFP Plasmids—Plasmid pJAH35-PAS2 was obtained by cloning the were generated as described by McCollum et al. (1993) and Monosov et 1.8-kb genomic DNA fragment containing the PpPAS2 gene as a SmaI al. (1996), respectively. PpPAS2, a Gene Involved in Peroxisomal Biogenesis 18975 FIG.1—continued sions in all peroxisomal subfractions were analyzed by SDS-PAGE and immunoblotting. Microscopy and Immunocytochemistry—GFP fluorescence in living cells was analyzed using the fluorescein isothiocyanate channel of a Photomicroscope III fluorescence microscope (Zeiss, Oberkochen, Ger- many) equipped with a Planopochromat 63/1.4 (oil) objective. Cells for electron microscopy were fixed with 1.5% w/v KMnO for 20 min at room temperature, washed extensively with water, resuspended in 0.5% w/v uranyl acetate, and incubated for 15 min at room temper- FIG.1. Nucleotide sequence of the PpPAS2 gene and the de- ature. Samples were embedded in Epon 812 after dehydration in a duced amino acid sequence. Shown is the translation of the PpPAS2 graded ethanol series. Ultrathin sections were examined in a Philips sequence from the ATG, at nucleotide 11, to the termination codon 300 electron microscope. (STOP), at nucleotides 1366–1368. The amino acids are in the one- Cells for immunocytochemistry were fixed in 3% v/v glutaraldehyde, letter code. Relevant restriction sites are indicated. 0.1 M sodium cacodylate, pH 7.2 for 30 min at 0 °C, dehydrated in a graded ethanol series, and embedded in Lowicryl K4 M. Immunolabel- Preparation of Crude Yeast Lysates—Yeast cells were harvested by ing was performed on ultrathin sections by the protein A-gold method centrifugation, resuspended in 3 volumes (compared with the volume of (Slot and Geuze, 1984), using anti-ScFox3p and anti-PpPas2p as pri- the pellet) of disruption buffer (20 mM Tris-Cl, pH 7.9, 10 mM MgCl ,1 mary antibodies and goat anti-rabbit immunoglobulin conjugated to mM EDTA, 5% v/v glycerol, 1 mM dithiothreitol, 0.3 M ammonium colloidal gold as secondary antibodies. Sections were poststained in sulfate, 0.2 mM phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin, and 5 0.5% w/v uranyl acetate and examined in a Philips 300 or CM10 elec- mg/ml aprotinin) and mixed with 4 volumes of acid-washed beads. Cell tron microscope. suspensions were vortexed for 1 min after which the tubes were placed on ice for 2 min. This treatment was repeated five times after which the RESULTS supernatants were removed. The beads were washed once with disrup- Cloning of PpPAS2 by Functional Complementation—Three tion buffer, and supernatants, representing the crude cell extracts, were Pppas2 mutants were isolated, selecting for cells deficient in pooled and stored at 220 °C. Subcellular Fractionations—Strains PPY4 (wild type for PpPAS2) peroxisome assembly (Gould et al., 1992), which were unable to and SEW1 (Pppas2 null mutant) were used for cell fractionation exper- grow on media containing oleic acid or methanol as sole carbon iments. Cells were cultured in 500 ml of YPD medium to near satura- sources. However, their growth was normal on glucose and tion, pelleted, resuspended in 2 liters of SM medium, and incubated for nonfermentable carbon sources such as glycerol, malate, etha- 24 h. Preparation of spheroplasts and cell homogenates, as well as the nol, and lactate. Through screening of a genomic DNA library, subsequent differential and density gradient centrifugation, were per- a 1.8-kb DNA fragment was obtained, which restored the abil- formed according to Monosov et al. (1996). Enriched organelle fractions (27,000 3 g pellet) of strains STW1 and ity of the mutant strain to utilize methanol and oleate. Se- SKF1, grown on SM medium, were diluted 10-fold either in 10 mM quencing of this DNA fragment revealed a 1,365-base pair open Tris-Cl, pH 8.5, or 1 M NaCl in 10 mM Tris-Cl, pH 8.5, or 0.1 M Na CO , 2 3 reading frame encoding a 455-amino acid protein (calculated pH 11.5, and incubated on ice for 1 h. Insoluble materials were pelleted molecular mass of 51,969 Da) with an estimated pI of 4.8 by centrifugation for1hat 100,000 3 g at 4 °C in a Beckman SW 50.1 (Fig. 1). rotor. Pellet fractions were resuspended in the appropriate buffer; after Deletion of PpPAS2 Abolishes Import of PTS1- and PTS2- centrifugation as above, the corresponding supernatants were pooled. Distributions of PpPas2p, PpPas8p, PpPer6p, catalase, and GFP fu- containing Reporter Proteins—The PpPAS2 gene was replaced 18976 PpPAS2, a Gene Involved in Peroxisomal Biogenesis FIG.2. Schematic representation of the PpPAS2 knockout construct. The gray bar represents the genomic fragment cloned by functional complementation of the Pppas2 mutant. The black bar marks the location of the PpPAS2 open reading frame. The PpPAS2 fragment that is replaced with the PpARG4 gene is indicated. Arrows indicate the direction of transcription. Ac, AccI; As, AseI; B, BamHI; E, EcoRI; H, HindII; HIII, HindIII; P, PstI; mcs, multiple cloning site of pBSII KS; bp, base pairs. FIG.4. Detection of GFP-PTS1 and PTS2-GFP in wild type and null mutant cells. The subcellular localization of the GFP-PTS1 and GFP-PTS2 reporter proteins was examined in living cells by fluores- cence microscopy. Wild type strain STW1 (panel A) and mutant strain FIG.3. Synthesis of PpPas2p in wild type and the null mutant. SEW2 (panel C), both expressing GFP-PTS1, were induced for 20 h in Crude extracts (100 mg of total protein) from wild type PPY1 cultured in methanol medium. Wild type strain STW2 (panel B) and mutant strain YPD (lane 1), YPM (lane 2), and the null mutant induced in YPM (lane SEW3 (panel D), both expressing PTS2-GFP, were induced for 20 h in 3) were subjected to SDS-PAGE. The proteins were transferred to oleate medium. Bar 5 1 mm. nitrocellulose and the blot incubated with anti-PpPas2p (dilution 1:5,000). Molecular mass markers are indicated on the right. The arrow marks PpPas2p, migrating at 52 kDa. as well as PTS2-GFP (Fig. 4D) were present in the cytoplasm as indicated by the diffuse fluorescent signal. with the PpARG4 gene (Fig. 2). The resulting Pppas2 null Peroxisomal Remnants Are Not Detectable in the Pppas2 Null mutant showed normal growth on glucose but was unable to Mutant—Ultrastructural examination of the null mutant did metabolize methanol and oleate, as observed for the original not reveal any morphologically distinguishable peroxisomes on mutants. In a genetic cross between the PpPas2 null mutant methanol (Fig. 5C) or oleate (results not shown). In wild type and the PpPas2 mutant, no complementation was observed for cells, the peroxisomal compartment was clearly visible and had growth on methanol or oleate. This indicates that the identified a characteristic morphology when grown in methanol (Fig. 5A) open reading frame was not that of a suppressor gene. Rein- and oleic acid (Fig. 5B). Large, clustered peroxisomes filled troduction of PpPAS2 into the null mutant restored the ability with alcohol oxidase were seen in methanol-grown cells; and to grow on methanol and oleate (results not shown). Notably, small, more abundant peroxisomes, identified as such by the the cloned 59-noncoding region of only 90 nucleotides was suf- presence of 3-ketoacyl-CoA thiolase, were evident in oleate- ficient to direct expression of the PpPAS2 gene on both oleate grown cells. and methanol (see below). An enriched organelle fraction was prepared from wild type Antibodies raised against PpPas2p specifically detected a cells induced on methanol by differential centrifugation and protein of approximately 52 kDa in lysates of wild type P. subjected to isopycnic centrifugation on a continous Nycodenz pastoris cells grown on YPD (Fig. 3, lane 1), and only a mod- gradient. Peroxisomes migrated to their buoyant density of erate 3–5-fold induction of the PpPas2p was noted on methanol 1.23 g/ml (fractions 2–6) as judged by the distribution of cata- (Fig. 3, lane 2) and oleate (results not shown). As expected, no lase (Fig. 6A), well separated from the bulk of mitochondria PpPas2p was detected in lysates from the null mutant (Fig. 3, (fractions 12–20) identified by cytochrome c oxidase. Or- lane 3). ganelles from the null mutant, fractionated in the same way, The import of peroxisomal matrix proteins was assessed by lacked normal peroxisomes, whereas mitochondria were found determining the intracellular location of GFP fused either to a at their normal position in the gradient (Fig. 6C). Only a small PTS1 or PTS2 sequence. A GFP-PTS1 fusion protein was ex- amount of catalase activity was recovered in organellar frac- pressed in strains STW1 (producing PpPas2p and GFP-PTS1) tions of lower density in the Nycodenz gradient. and SEW2 (producing GFP-PTS1 but lacking PpPas2p). Upon Equal portions of the gradient fractions from both wild type induction on methanol, the GFP-PTS1 protein was directed to and null mutant cells were analyzed by immunoblotting. Alco- the peroxisomes of STW1 cells as judged by the intense fluo- hol oxidase, PpPas8p, and PpPas2p were predominantly per- rescent spots representing the large clustered peroxisomes oxisomal (Fig. 6B), with some trailing of PpPas2p and PpPas8p (Fig. 4A). These results are in agreement with data published into the lighter parts of the gradient, a phenomenon observed by Monosov et al. (1996). Likewise, a hybrid protein consisting frequently with membrane-associated proteins. In the gradient of the NH -terminal 16 amino acids of S. cerevisiae thiolase and from the null mutant, none of these proteins were detected in GFP (PTS2-GFP) was targeted to the peroxisomes in oleate- the denser parts of the gradient where peroxisomes usually grown STW2 (expressing PpPas2p and PTS2-GFP) (Fig. 4B). band (Fig. 6, C and D). However, as observed in the wild type, This implies that the sequence identified as the PTS for thio- traces of PpPas8p were found in gradient fractions of lower lase is also properly recognized as such in P. pastoris. In strains density in the null mutant (Fig. 6, B and D). SEW2 and SEW3, lacking PpPas2p, both GFP-PTS1 (Fig. 4C) PpPas2p Is a Peroxisomal Membrane Protein—The location PpPAS2, a Gene Involved in Peroxisomal Biogenesis 18977 FIG.5. Ultrastructural analysis of wild type and null mutant cells. Sec- tions of KMnO -fixed, methanol-grown cells of wild type PPY1 (panel A) and the null mutant (panel C) are shown. Panel B, immunocytochemical detection of the b-oxidation enzyme 3-ketoacyl-CoA thio- lase using anti-ScFox3p in a section of p-formaldehyde-fixed, oleic acid-grown cells of PPY1. Note that the morphologi- cal appearance of the peroxisomal com- partment differs, with a few large clus- tered peroxisomes in methanol-induced cells (panel A) and smaller, more abun- dant, and dispersed peroxisomes in oleate-induced cells (panel B). No peroxi- somal structures are visible in the null mutant (panel C). The vesicles adjacent to the nuclear membrane are also observed routinely in wild type P. pastoris cells (Gould et al., 1992) and are not thought to be of peroxisomal origin. Panels D and E, immunocytochemical detection of PpPas2p in methanol-grown cells using anti-PpPas2p or the appropriate preim- mune serum as the control (panel F). N, nucleus; V, vacuole; M, mitochondrion. Bar, 0.5 mm. of PpPas2p within the peroxisomes was analyzed by immuno- erly targeted to peroxisomes. cytochemistry and organelle subfractionation. Sections of To address the subperoxisomal localization of the 1–40-GFP methanol-grown wild type cells were incubated with anti- fusion, crude organellar fractions were prepared from strains PpPas2p followed by gold-conjugated protein A. The gold par- STW1 and SKF1 grown on SM medium, and the extractability ticles almost exclusively decorated the peroxisomal membrane of the fusion proteins after low salt, high salt, or carbonate (Fig. 5, D and E). A control incubation using the preimmune treatment was analyzed by Western blotting. In strain STW1, antiserum showed no labeling of the peroxisomal membrane the GFP-PTS1 fusion protein was completely soluble after car- (Fig. 5F). bonate treatment, and even after low and high salt extraction Strain STW1 was grown in SM medium, and crude organelle the bulk of the fusion protein was found in the supernatants as pellets were fractionated into soluble and insoluble fractions expected for soluble matrix proteins (Fig. 7A). In strain SKF1, after treatment with 10 mM Tris, pH 8.5 (low salt), 1 M NaCl in however, the 1–40-GFP fusion protein was insoluble under all three conditions (Fig. 7B), behaving like the full-length 10 mM Tris, pH 8.5 (high salt), or 0.1 M Na CO as described 2 3 under “Materials and Methods.” PpPas2p, PpPas8p, and PpPas2p, as well as PpPas8p and PpPer6p (Fig. 7A). PpPer6p were insoluble under all three conditions (Fig. 7A), PpPas2p Is Homologous to S. cerevisiae Pas3p Protein—A thus behaving like integral membrane proteins. Catalase, a data base search using the BLAST program revealed that the marker for soluble matrix proteins, was found in the superna- PpPas2p protein displays a high similarity to ScPas3p, a per- tant under all conditions tested. oxisomal integral membrane protein (Ho¨hfeld et al., 1991) but The NH -terminal 40 Amino Acids of PpPas2p Contain a not to other proteins. From the sequence alignment (Fig. 9A)an Peroxisomal Targeting Signal—Analysis of the PpPas2p amino overall identity of 35% was determined. There is a significant acid sequence did not reveal any known PTS. Several NH - similarity (62%) over the entire length of PpPas2p and terminal fragments of PpPAS2p, varying in length from 40 to ScPas3p, but a number of segments that are highly conserved 110 amino acids, were tested for their ability to target the can be distinguished (e.g. PpPas2p amino acid residues 49–64, reporter protein GFP to peroxisomes. As shown in Fig. 8, the 70–78, 215–233, and 442–453). The putative membrane-span- smallest hybrid protein, consisting of the NH -terminal 40 ning domain (residues 18–39) and membrane-anchoring do- amino acids of PpPas2p fused to GFP (1–40-GFP), was prop- main (residues 135–153) of ScPas3p do not seem to be partic- 18978 PpPAS2, a Gene Involved in Peroxisomal Biogenesis FIG.7. Subperoxisomal localization of PpPas2p and heterolo- gous proteins in strains STW1 (GFP-PTS1) and SKF1 (1–40- GFP). Crude organelle fractions were prepared from methanol-induced strains STW1 (panel A) and SKF1 (panel B) and subfractionated into an insoluble pellet fraction (P) and a soluble fraction (S) after treatment with NaCl, Tris, or Na CO (Carb.) as described under “Materials and 2 3 Methods.” Distributions of PpPas2p, PpPas8p, PpPer6p, catalase, and the GFP fusion proteins between supernatant and membranous pellet fractions were examined by immunoblotting. The distribution of the four endogenous proteins was identical for both strains and is shown for strain STW1. Antibodies used were a-PpPas2p (dilution 1:5,000), a-PpPas8p (dilution 1:10,000), a-PpPer6p (dilution 1:2,000), a-catalase (dilution 1:5,000), and a-GFP (dilution 1:5,000). FIG.6. Nycodenz density gradient fractionation of organelles from wild type and null mutant cells. Crude organelle fractions FIG.8. A PTS resides near the NH terminus of PpPAS2p. from methanol-induced cells, consisting primarily of peroxisomes and Localization of a hybrid protein consisting of the NH -terminal 40 mitochondria, were fractionated on Nycodenz gradients as described amino acids of PpPAS2p and the GFP (1–40-GFP) was determined by under “Materials and Methods.” The distribution of peroxisomal cata- confocal microscopy. P. pastoris cells producing the hybrid protein were lase and mitochondrial cytochrome c oxidase in gradients of wild type grown in synthetic media containing methanol as sole carbon source. strain PPY4 (panel A) and null mutant SEW1 (panel C) is shown. Note that the cells have large, clustered peroxisomes (panel A) which Enzyme units are expressed as relative specific activities, reflecting the colocalize with the GFP fluorescence (panel B). percentage enzyme activity in a given fraction relative to the total amount of protein contained in the gradient. Equal volumes of fractions 1995; Liu et al., 1995) reminiscent of the structures seen in from the gradients of wild type PPY4 (panel B) and the null mutant SEW1 (panel D) were analyzed by immunoblotting. Blots were incu- human patients suffering from generalized peroxisomal disor- bated with antisera against PpPas8p (a-PpPas8p, dilution 1:5,000), ders. The Pppas2 null mutant described in this study is an PpPas2p (a-PpPas2p, dilution 1:5,000), and against alcohol oxidase exception to this rule since it lacks morphologically detectable (a-PpAox1p, dilution 1:20,000). No PpAox1p could be detected in gra- peroxisomes (Fig. 5C) even upon induction on methanol or dient fractions of the null mutant. oleate, both of which are known to induce peroxisomes. In cell fractionation experiments normal peroxisomes could not be ularly conserved in PpPas2p. The hydrophobicity plot, detected either (Fig. 6, C and D). Traces of the peroxisomal prepared according to Kyte and Doolittle (1982), shows that the membrane-associated protein PpPas8p, however, were found NH -terminal portion of PpPas2p is not as hydrophobic as the in fractions of lower density near the top of the gradient in both corresponding part of ScPas3p (Fig. 9B, compare with Fig. 5. in wild type (Fig. 6B) and the null mutant (Fig. 6D). Whether this Ho¨hfeld et al. (1991)). However, two hydrophobic domains seem represents a real peroxisomal (precursor) fraction or nonspe- to be formed by amino acid residues 66–79 and 149–174. cific binding of PpPas8p to mitochondria or other membranous DISCUSSION particles is unknown. In the Pppas2 null mutant, marker pro- Most of the P. pastoris pas and per mutants characterized to teins containing a PTS1 or a PTS2 sequence were found to be date harbor morphologically and biochemically detectable per- localized to the cytoplasm (Fig. 4). These data suggest that oxisome ghosts or membrane remnants (McCollum et al., 1993; PpPas2p is involved in the early stages of peroxisome biogen- Spong and Subramani, 1993; Heyman et al., 1994; Kalish et al., esis. Therefore, we propose that the proper functioning of PpPAS2, a Gene Involved in Peroxisomal Biogenesis 18979 PpPas2p is a prerequisite for the assembly of matrix, and at least some membrane, proteins into peroxisomes. PpPas2p is homologous to Pas3p of S. cerevisiae (ScPas3p), which has been shown by Ho¨hfeld et al. (1991) to be a peroxi- somal integral membrane protein. Immunocytochemical detec- tion of PpPas2p revealed that it is also associated with the peroxisomal membrane (Fig. 5, D and E). This was further substantiated by biochemical experiments that showed that the protein behaves like an integral membrane protein during subfractionation of an organelle pellet fraction (Fig. 7). Earlier experiments show that there are at least two differ- ent signals that target proteins to the peroxisomal matrix. PMPs must use a different targeting signal, which remains to be defined. Proteins that reside in the peroxisomal membrane can be envisaged as having two components: an mPTS, which targets the protein to the peroxisome, and a transmembrane domain (TMD), which anchors the protein in the membrane, or a protein-protein interaction domain that anchors it to the membrane through other peroxisomal integral membrane pro- teins. These two components could either be separate or overlapping. The PTS in ScPas3p has been described to lie in the NH - terminal half of the protein, a region that encompasses a pu- tative transmembrane domain (Ho¨hfeld et al., 1992). Our result on the targeting of the 1–40-GFP fusion reveals the presence of an mPTS in the NH -terminal 40 amino acids of PpPas2p (Fig. 8). In contrast to ScPas3p, the first 40 amino acids of PpPas2p do not contain any obvious TMD (Figs. 1 and 9B). Yet the 1–40-GFP fusion is directed faithfully to the peroxisomal mem- brane, where it behaves exactly like the full-length PpPas2p in terms of its inextractability with sodium carbonate (Fig. 7). This suggests that these 40 amino acids have both an mPTS as well as a domain that allows the fusion to interact tightly with the peroxisomal membrane or with one or more PMPs. The sequence and experimental data seem contradictory, having a protein behaving like an integral membrane protein without predicted transmembrane segments. On one hand, several (peroxisomal) proteins have been shown to be sodium carbon- ate-inextractable (Tan et al., 1995; Erdmann and Blobel, 1995) from the membranes, whereas no clear transmembrane seg- ments could be detected by sequence analysis. On the other hand, some proteins with strong protein-protein interactions might resist the sodium carbonate extraction procedure and therefore are falsely classified as integral membrane proteins. Based on the relatively low content of hydrophobic residues and the presence of a highly positively charged domain in the FIG.9. Sequence comparison and hydrophobicity analysis of NH -terminal 40 amino acids, we favor the possibility of a PpPas2p and ScPas3p. The amino acid sequences were aligned using strong interaction between this sequence in PpPas2p and an- the BESTFIT program (panel A). Identical residues are indicated by an other peroxisomal integral membrane protein. asterisk below the sequence; a dot represents aligned residues that are Based on the proposed topology of ScPAS3p, we expected similar. Two hydrophobic domains, identified by Ho¨hfeld et al. (1991), that the GFP segment in the 1–40-GFP hybrid protein would in ScPas3p are underlined. Similarity rules: G 5 A 5 S; V 5 I 5 L 5 M 5 F 5 Y 5 W; K 5 r 5 H; D 5 E 5 Q 5 N; S 5 T 5 Q 5 N. Panel face the cytosol. Therefore, we performed a proteinase K pro- B, hydrophobicity plot of PpPas2p and ScPas3p according to Kyte and tection experiment on a crude organellar fraction to determine Doolittle (1982), using a window size of 19 amino acids. Two hydropho- whether it would be sensitive to proteolytic degradation. The bic domains are revealed in PpPas2p, a 13-amino acid stretch at posi- tion 66–79, and a potential membrane-spanning region at position hybrid protein appeared to be resistant to proteolytic degrada- 149–174. tion, whether or not the organelles were disrupted by Triton X-100 prior to the proteinase K treatment. This suggests that GFP itself is highly resistant to proteolytic degradation, and 1994). In a more detailed analysis of the targeting information therefore the results were not conclusive. in CbPMP47 it was shown that a stretch of 20 amino acids in A putative mPTS has been identified in a Candida boidinii the intervening loop between transmembrane domains 4 and 5 protein, CbPMP47. This protein is homologous to a family of was able to direct a soluble reporter protein to the peroxisomal mitochondrial solute transporters that span the membrane six membrane of S. cerevisiae (McNew and Goodman, 1996). It is times (Kuan and Saier, 1993). The targeting information on interesting to note that in both PpPas2p and in CbPMP47 the this protein was localized to a region of the protein containing mPTS does not include any predicted TMD. This supports the transmembrane domains 4 and 5 and an intervening 20-amino idea proposed above that the mPTS and TMD are separable acid loop facing the peroxisomal matrix (McCammon et al., entities and that it is the mPTS that directs a TMD in a protein 18980 PpPAS2, a Gene Involved in Peroxisomal Biogenesis Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–267 to the peroxisomal membrane. This model predicts that the Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, pp. 92–114, TMDs of PMPs would have no higher affinity for the peroxiso- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY mal membrane relative to other subcellular membranes. Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Nature 373, 663–664 Heyman, J. A., Monosov, E., and Subramani, S. (1994) J. Cell Biol. 127, 1259–1273 What are the features common to mPTSs from different Ho¨hfeld, J., Veenhuis, M., and Kunau, W. H. (1991) J. Cell Biol. 114, 1167–1178 proteins? A comparison of the 20-amino acid segment of Cb- Ho¨hfeld, J., Mertens, D., Wiebel, F. F., and Kunau, W.-H. (1992) in Membrane Biogenesis and Protein Targeting (Neupert, W., and Lill, R., eds) pp. 185–207, PMP47 and the NH termini of PpPas2p and ScPas3p reveals Elsevier, New York a block of five amino acids (of which four are positively charged) Imanaka, T., Shiina, Y., Takano, T., Hashimoto, T., and Osumi, T. (1996) J. Biol. in CbPMP47 which is also observed in both PpPas2p and Chem. 271, 3706–3713 Kalish, J. E., Theda, C., Morrell, J. C., Berg, J. M., and Gould, S. J. (1995) Mol. ScPas3p (QIKRR) at positions 53–57 and 59–63, respectively, Cell. Biol. 15, 6406–6419 being 100% conserved between these Pas proteins. Notably, Keller, G. A., Krisans, S. K., Gould, S. J., Sommer, J. M., Wang, C. C., Schliebs, W., this sequence is excluded from the first 40 amino acids of Kunau, W.-H., Brody, S., and Subramani, S. (1991) J. Cell Biol. 114, 893–904 Kuan, J., and Saier, M. J. (1993) Res. Microbiol. 144, 671–672 PpPas2p, which are able to direct GFP to the peroxisomal Kunau, W.-H, Kionka, C., Ledebur, A., Mateblowski, M., Moreno de la Garza, M., membrane. Another positively charged sequence (RRNKKK) Schultz-Borchard, U., Thieringer, R., and Veenhuis, M. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 128–140, Springer present at position 10–15 might be involved in targeting of Verlag, Berlin PpPas2p, although this sequence is not particularly conserved Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105–132 in ScPas3p. Laemmli, U. K. (1970) Nature 227, 680–685 Lazarow, P. B., and Moser, H. W. (1989) in The Metabolic Bases of Inherited Mutational analysis of the first 40 amino acids of PpPas2p Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 6th Edition, will reveal whether the mPTS and peroxisomal membrane pp. 1479–1509, McGraw Hill, New York Liu, H., Tan, X., Veenhuis, M., McCollum, D., and Cregg, J. M. (1992) J. Bacteriol. interaction domain are overlapping or physically separable 174, 4943–4951 entities. It may also help address whether an mPTS and per- Liu, H., Tan, X., Russell, K. A., Veenhuis, M., and Cregg, J. M. (1995) J. Biol. oxisomal membrane interaction domain need to coexist for Chem. 270, 10940–10951 Madden, E. A., and Storrie, B. (1987) Anal. Biochem. 163, 350–357 proper targeting to the peroxisomal membrane. Marzioch, M., Erdmann, R., Veenhuis, M., and Kunau, W.-H. (1994) EMBO J. 13, Studies on the import of PMPs into peroxisomes in vitro have 4908–4917 been undertaken without any knowledge of the mPTSs in- McCammon, M. T., McNew, J. A., Willy, P. J., and Goodman, J. M. (1994) J. Cell Biol. 124, 915–925 volved in the targeting process (Diestelkotter and Just, 1993; McCollum, D., Monosov, E., and Subramani, S. (1993) J. Cell Biol. 121, 761–774 Imanaka et al., 1996). These experiments reveal that import of McNew, J. A., and Goodman, J. M. (1996) Trends Biochem. Sci. 21, 54–58 Monosov, E. Z., Wenzel, T. J., Lu¨ers, G. H., Heyman, J. A., and Subramani, S. such proteins is dependent on time and temperature, does not (1996) J. Histochem. Cytochem. 44, 581–589 require ATP or GTP, and is not inhibited by N-ethylmaleimide Motley, A., Hettema, E., Distel, B., and Tabak, H. F. (1994) J. Cell Biol. 125, treatment of either the soluble components or of the isolated 755–767 Nuttley, W. M., Brade, A. M., Gaillardin, C., Eitzen, G. A., Glover, J. R., peroxisomes. In contrast, the import of the peroxisomal matrix Aitchinson, J. D., and Rachubinski, R. A. (1993) Yeast 9, 507–517 marker, firefly luciferase, into peroxisomes of permeabilized Osumi, T., Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikta, M., cells is dependent on ATP hydrolysis and is blocked by N- Miyazawa, S., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 181, 947–954 ethylmaleimide pretreatment of the cytosol-depleted cells Rapp, S., Soto, U., and Just, W. W. (1993) Exp. Cell Res. 205, 59–65 (Rapp et al., 1993; Wendland and Subramani, 1993). These Rickey, C. (1990) Mol. Biol. Rep. 9, 2–3 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, properties, as well as the import of PMPs into peroxisome 5463–5467 ghosts in yeast and human cell lines deficient in the import of Slawecki, M. L., Dodt, G., Steinberg, S., Moser, A. B., Moser, H. W., and Gould, S. PTS1- and PTS2-containing proteins, imply that the mecha- J. (1995) J. Cell Sci. 108, 1817–1829 Slot, J. W., and Geuze, H. J. 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Published: Aug 1, 1996

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