Title: AtPex14p maintains peroxisomal functions by determining protein targeting to three kinds of plant peroxisomes
Abstract: Article1 November 2000free access AtPex14p maintains peroxisomal functions by determining protein targeting to three kinds of plant peroxisomes Makoto Hayashi Makoto Hayashi Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan Search for more papers by this author Kazumasa Nito Kazumasa Nito Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies, Okazaki, 444-8585 Japan Search for more papers by this author Kanako Toriyama-Kato Kanako Toriyama-Kato Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Maki Kondo Maki Kondo Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Tomoyuki Yamaya Tomoyuki Yamaya Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan Search for more papers by this author Mikio Nishimura Corresponding Author Mikio Nishimura Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies, Okazaki, 444-8585 Japan Search for more papers by this author Makoto Hayashi Makoto Hayashi Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan Search for more papers by this author Kazumasa Nito Kazumasa Nito Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies, Okazaki, 444-8585 Japan Search for more papers by this author Kanako Toriyama-Kato Kanako Toriyama-Kato Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Maki Kondo Maki Kondo Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Search for more papers by this author Tomoyuki Yamaya Tomoyuki Yamaya Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan Search for more papers by this author Mikio Nishimura Corresponding Author Mikio Nishimura Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies, Okazaki, 444-8585 Japan Search for more papers by this author Author Information Makoto Hayashi1,2, Kazumasa Nito1,3, Kanako Toriyama-Kato1, Maki Kondo1, Tomoyuki Yamaya2 and Mikio Nishimura 1,3 1Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan 2Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan 3Department of Molecular Biomechanics, School of Life Science, Graduate University of Advanced Studies, Okazaki, 444-8585 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5701-5710https://doi.org/10.1093/emboj/19.21.5701 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We previously isolated an Arabidopsis peroxisome-deficient ped2 mutant by its resistance to 2,4-dichlorophenoxybutyric acid. Here, we describe the isolation of a gene responsible for this deficiency, called the PED2 gene, by positional cloning and confirmed its identity by complementation analysis. The amino acid sequence of the predicted protein product is similar to that of human Pex14p, which is a key component of the peroxisomal protein import machinery. Therefore, we decided to call it AtPex14p. Analyses of the ped2 mutant revealed that AtPex14p controls intracellular transport of both peroxisome targeting signal (PTS)1- and PTS2-containing proteins into three different types of peroxisomes, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes. Mutation in the PED2 gene results in reduction of enzymes in all of these functionally differentiated peroxisomes. The reduction in these enzymes induces pleiotropic defects, such as fatty acid degradation, photorespiration and the morphology of peroxisomes. These data suggest that the AtPex14p has a common role in maintaining physiological functions of each of these three kinds of plant peroxisomes by determining peroxisomal protein targeting. Introduction Peroxisomes in higher plant cells are known to differentiate into at least three different classes, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes (Beevers, 1979). Each organelle contains a unique set of enzymes that provides special functions in various organs in higher plants. Glyoxysomes are present in cells of storage organs, such as endosperms and cotyledons during post-germinative growth of oil-seed plants, as well as in senescent organs (Nishimura et al., 1996). They contain enzymes for fatty acid β-oxidation and the glyoxylate cycle, and play a pivotal role in the conversion of lipid into sucrose. It has been suggested that fatty acids are exclusively degraded in glyoxysomes (i.e. not in mitochondria) during germination and post-germinative growth (Beevers, 1982). In contrast, leaf peroxisomes are found widely in cells of photosynthetic organs. It has been shown that some of the enzymes responsible for photorespiration are localized in leaf peroxisomes even though the entire photorespiratory process involves a combination of enzymic reactions that occur in chloroplasts, leaf peroxisomes and mitochondria (Tolbert, 1982). Other organs, such as roots and stems, contain unspecialized peroxisomes whose function is still obscure (Nishimura et al., 1996). Glyoxysomes, leaf peroxisomes and unspecialized peroxisomes are known to be converted into one another under certain conditions (Nishimura et al., 1996). For example, glyoxysomes in etiolated cotyledons are transformed directly into leaf peroxisomes during the greening of cotyledons (Titus and Becker, 1985; Nishimura et al., 1986). During this process, glyoxysomal enzymes, such as malate synthase, are specifically degraded (Mori and Nishimura, 1989), and leaf peroxisomal enzymes, such as glycolate oxidase and hydroxypyruvate reductase, are newly synthesized and transported into the organelle as it is being transformed from a glyoxysome to a leaf peroxisome (Tsugeki et al., 1993; Hayashi et al., 1996b). Leaf peroxisomes in green cotyledons are subsequently converted to glyoxysomes when the cotyledons undergo senescence (De Bellis and Nishimura, 1991; Nishimura et al., 1993). It has been suggested that the functional transformation of plant peroxisomes is controlled by gene expression, protein translocation and protein degradation, although the detailed mechanisms underlying these processes still need to be clarified (Nishimura et al., 1996). To identify the genes responsible for regulation of peroxisomal function in plant cells, we isolated mutants with defective peroxisomes. To screen such mutants, we used 2,4-dichlorophenoxybutyric acid (2,4-DB) as a compound for detecting Arabidopsis mutants with defects in glyoxysomal fatty acid β-oxidation (Hayashi et al., 1998). We expected that two methylene groups of the butyric side chain in 2,4-DB would be removed by the action of glyoxysomal fatty acid β-oxidation to produce a herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in wild-type plants, whereas the mutants no longer produce a toxic level of 2,4-D from 2,4-DB because of the defect in fatty acid β-oxidation. We succeeded in identifying four mutants that were classified as carrying alleles at three independent loci. We designated these loci as ped1, ped2 and ped3, respectively, where ped stands for peroxisome defective. These mutants required sucrose for post-germinative growth, because the reduced activity of glyoxysomal fatty acid β-oxidation prevented the production of sucrose from the lipid reserves in seeds. One of these mutants, ped2, has been demonstrated previously to have a defect in the intracellular transport of 3-ketoacyl CoA thiolase, an enzyme participating in fatty acid β-oxidation, from the cytosol to glyoxysomes (Hayashi et al., 1998). Peroxisomal enzymes are synthesized in the cytosol, and function after their post-translational transport into peroxisomes. Most of the plant peroxisomal enzymes have been shown to contain one of two peroxisome targeting signals (PTSs) within their amino acid sequences (Hayashi, 2000). One type of targeting signal (PTS1) is a unique tripeptide sequence found in the C-terminus of the proteins (Hayashi et al., 1996a; Trelease et al., 1996). The permissible combinations of tripeptide sequence for plant PTS1 are (C/A/S/P)-(K/R)-(I/L/M) (Hayashi et al., 1997). Another type of targeting signal is involved in a cleavable N-terminal presequence (Gietl et al., 1994). The N-terminal presequences contain a consensus sequence (R)-(L/Q/I)-X5-(H)-(L) (X stands for any amino acid) called PTS2 (Kato et al., 1996a, 1998). These proteins are synthesized as precursor proteins, which show a higher molecular mass due to the N-terminal presequence. The N-terminal presequence is processed to form the mature protein after its transport into peroxisomes. These peroxisomal proteins with PTS1 or PTS2 are imported into peroxisomes with oligomeric forms (Lee et al., 1997; Flynn et al., 1998; Kato et al., 1999). Here we report the identification and analysis of the PED2 gene, and present evidence that the gene product of PED2 is a component of the protein targeting machinery involved in each of the three kinds of plant peroxisome. We discuss the pleiotropic defects in the ped2 mutant based upon the predicted function of the PED2 gene product. Results High-resolution mapping of the PED2 locus The Arabidopsis ped2 mutant, which has a Landsberg erecta ecotype background, was identified by its resistance to the presence of 2,4-DB. This mutant requires sucrose for post-germinative growth, because of its reduced activity for fatty acid β-oxidation. Our initial mapping of PED2 located it to the lower arm of chromosome 5, between two molecular markers, LFY3 and g2368 (Hayashi et al., 1998). We outcrossed the ped2 mutant (which has a Landsberg erecta ecotype background) to wild-type Arabidopsis, which has a Columbia ecotype background, and identified 310 F2 progenies that have homozygous ped2 alleles for high-resolution mapping. These progenies were subsequently scored according to their genetic background at a series of molecular markers using the cleaved amplified polymorphic sequence (CAPS) mapping procedure described by Konieczny and Ausubel (1993) (Figure 1A). The number of chromosomes that showed a Columbia background represents the number of recombinations that occurred between the PED2 locus and the position of each molecular marker, since the genomic DNA of the ped2 mutant has a Landsberg erecta background. To identify the genetic background of chromosome 5 between LFY3 and g2368, we generated four molecular markers, MRG21-3, MQB2-1, MQB2-4, MHJ24-4, based on the nucleotide sequences of P1 contigs that have been reported by the Kazusa DNA Institute, Chiba, Japan (http://www.kazusa.or.jp/kaos/). As summarized in Figure 1A, high-resolution mapping revealed that the PED2 locus is located between MRG21-3 and MQB2-4. The closest molecular marker to the PED2 locus is MQB2-1. This result strongly suggests that the PED2 gene is contained within a single P1 clone, MQB2. Figure 1.Positional cloning of the PED2 gene. (A) High-resolution mapping of PED2 on chromosome 5. Names and positions of the molecular markers used in this study are indicated on the top of the illustration. Hatched bars represent the regions covered by the P1 clones. We analyzed 310 F2 progeny (620 chromosomes) having homozygous ped2 alleles. The numbers of recombinations that occurred between the PED2 locus and the molecular markers are indicated at the bottom of the illustration. Mapping results with a series of molecular markers between LFY3 and MHJ24-4 are summarized schematically and indicate that the PED2 locus may be located within a single P1 clone, MQB2. (B) Schematic diagram of a 7734 bp XhoI fragment that is involved in the P1 clone, MQB2. The 12 black bars represent protein coding regions determined from the cDNA sequence. The triangle on the sixth black bar indicates the position of a nonsense mutation that occurs in the ped2 mutant. Nucleotide residue 1 corresponds to an adenine of the first methionine codon. (C) Effects of 2,4-DB on the growth of transgenic ped2 seedlings [ped2(PED2)] harboring the 7734 bp XhoI fragment shown in (B). Wild-type Arabidopsis (WT), ped2 mutant (ped2) and ped2(PED2) were grown for 10 days on growth medium containing 0.2 μg/ml 2,4-DB under constant illumination. Photographs were taken after the seedlings were removed from the media and rearranged on agar plates. (D) Effects of sucrose on the growth of ped2(PED2) seedlings. Wild-type Arabidopsis (WT), ped2 mutant (ped2) and ped2(PED2) were grown for 10 days on growth medium without sucrose under constant illumination. Photographs were taken after the seedlings were removed from the media and rearranged on agar plates. Download figure Download PowerPoint Identification of the PED2 gene MQB2 is reported to contain 16 predicted genes (http://www.kazusa.or.jp/kaos/). Based on the nucleotide sequences, we designed a set of oligonucleotide primers that could amplify one of the predicted genes by using the PCR. This gene is located within the 7734 bp XhoI fragment contained in MQB2 (Figure 1B). DNA fragments were amplified from genomic DNAs of wild-type Arabidopsis (ecotype Landsberg erecta) and the ped2 mutant, using this primer set, and were fully sequenced. The nucleotide sequences of the two fragments are identical except for one nucleotide substitution, from C in the wild-type plant to T in the ped2 mutant (Figure 1B, arrowhead). This result strongly indicated that the 7734 bp XhoI fragment contained the PED2 gene. To confirm this result, the 7734 bp XhoI fragment isolated from the MQB2 clone was inserted into a plant binary vector, pBI121Δ35S, and then transformed into the ped2 mutant by Agrobacterium-mediated transformation (Bechtold et al., 1993). Seeds from individual kanamycin-resistant T2 progenies were scored for kanamycin resistance to identify the lines that are homozygous for the transgene. The homozygous T3 lines were assayed for 2,4-DB resistance and a sucrose requirement during post-germinative growth. As we have previously reported, the ped2 mutant was resistant to a toxic level of 2,4-DB, while it was sensitive to the absence of sucrose in the growth medium (Figure 1C and D, ped2). In contrast, the ped2 mutant transformed with the 7734 bp XhoI fragment became sensitive to a toxic level of 2,4-DB, whereas it was resistant to the absence of sucrose in the growth medium [Figure 1C and D, ped2(PED2)]. These phenotypes are identical to wild-type plants (Figure 1C and D, WT). These data indicated that the genomic sequence determined in this study corresponds to the PED2 gene. The nucleotide sequence data of PED2 are available in the DDBJ/EMBL/GenBank nucleotide sequence databases (AB037538). PED2 encodes a protein similar to Pex14p A cDNA clone of the PED2 gene was generated using RT–PCR with total RNA isolated from wild-type plants. The first methionine that appeared in this cDNA represents the start of the open reading frame, since the 5′ primer used for the PCR was designed to hybridize with the 5′ untranslated region including an in-frame stop codon. We determined the nucleotide sequence of the cDNA (DDBJ/EMBL/GenBank accession No. AB037539). Comparison of the cDNA and genomic DNA sequences showed that the PED2 gene contains 12 exons (Figure 1B). The deduced amino acid sequence of the gene product is composed of 507 amino acid residues (Figure 2). A nucleotide substitution occurred in the ped2 mutant, converting a CAA codon encoding Gln254 of the gene product to a stop codon (TAA) (Figure 2, asterisk). The amino acid sequence of the gene product shows significant similarity to mammalian and fungal Pex14p, one of the peroxisomal membrane proteins involved in the peroxisomal protein targeting machinery (Albertini et al., 1997; Brocard et al., 1997; Komori et al., 1997; Shimizu et al., 1999; Will et al., 1999), and is most similar to that of human Pex14p (Figure 2). Therefore we decided to call it AtPex14p. AtPex14p contains at lease two hydrophobic segments and a coiled-coil region (Figure 2). Although two yeast Pex14p are known to contain the class II SH3 ligand consensus sequence (Albertini et al., 1997), AtPex14p does not contain such a motif. In addition, there is no obvious PTS. Figure 2.Alignment of amino acid sequences for the PED2 gene product with mammalian and yeast Pex14p. Deduced amino acid sequence of the PED2 gene product (AtPex14p) was compared with Pex14p identified from human (HsPex14p), rat (RnPex14p), H.polymorpha (HpPex14p) and Saccharomyces cerevisiae (ScPex14p). Identical amino acid residues between AtPex14p and other Pex14p are highlighted. Amino acid sequence of AtPex14p is 29.6% identical to that of human Pex14p. The asterisk on Gln254 represents the position of a nonsense mutation (CAA to TAA) in the ped2 gene. Two putative membrane spanning domains are indicated by a line. A dashed line represents a putative coiled-coil region. Download figure Download PowerPoint Subcellular localization of AtPex14p To analyze the subcellular localization of AtPex14p, we prepared an antiserum raised against a fusion protein containing a partial amino acid sequence of AtPex14p (Met1–Pro100). This antiserum recognized a 75 kDa protein in wild-type plant (Figure 3, WT), while no cross-reactive band was detected in the ped2 mutant (Figure 3, ped2). These data indicate that AtPex14p is the 75 kDa protein. Figure 3.Immunodetection of AtPex14p in etiolated cotyledons of wild-type Arabidopsis and ped2 mutant. Extracts were prepared from 5-day-old etiolated cotyledons of wild-type Arabidopsis (WT) and ped2 mutant (ped2). For each sample, 10 μg of total protein were subjected to SDS–PAGE. Immunoblot analysis was performed using the antibody raised against AtPex14p. Markers are shown on the right with molecular masses in kDa. Download figure Download PowerPoint To investigate the subcellular localization of Pex14p in plant cells, homogenates prepared from pumpkin etiolated cotyledons were subjected to sucrose density gradient centrifugation. Fractions thus obtained were analyzed using an immunoblotting technique with the antibody raised against AtPex14p (Figure 4A). The 75 kDa protein was detected in fractions 23–25, whose densities were 1.25 g/cm3. These fractions also contained other glyoxysomal marker enzymes, such as isocitrate lyase and catalase, while they did not contain a mitochondrial marker enzyme (cytochrome c oxidase) activity (Figure 4A and B). Figure 4.Subcellular localization of Pex14p in pumpkin etiolated cotyledons. (A) Subcellular fractionation of etiolated pumpkin cotyledons was performed by 30–60% sucrose density gradient centrifugation. Fraction number 1 represents the top fraction of the gradient. Pex14p and isocitrate lyase in each fraction were detected by immunoblot analyses using antibody raised against AtPex14p (AtPex14p) and isocitrate lyase (ICL). (B) Sucrose concentration (open circles), activities of catalase (filled circles) and cytochrome c oxidase (COX; filled triangles) in the same fractions as used in (A) were also measured. (C) Intact glyoxysomes were resuspended in either low salt buffer (L), high salt buffer (H) or alkaline solution (A). Each buffer consists of 10 mM HEPES–KOH pH 7.2, 500 mM KCl with 10 mM HEPES–KOH pH 7.2 and 0.1 M Na2CO3, respectively. These samples were then centrifuged and separated into soluble (S) and insoluble (P) fractions. T represents total proteins of the intact glyoxysomes. Pex14p (AtPex14p), peroxisomal ascorbate peroxidase (pAPX) and isocitrate lyase (ICL) were detected by immunoblot analysis. (D) The intact glyoxysomes were treated with various concentrations of proteinase K in the absence (−) or presence (+) of Triton X-100. The concentration of proteinase K is indicated in μg/ml on the top of each lane. Pex14p (AtPex14p) and isocitrate lyase (ICL) were detected by immunoblot analysis. Download figure Download PowerPoint Figure 4C represents the result of the extensive subfractionation studies performed by the treatment of intact glyoxysomes with various solutions. Pex14p and ascorbate peroxidase, a marker enzyme for peroxisomal membranes (Yamaguchi et al., 1995), were found in the insoluble fraction even after treatment with alkaline solution. However, isocitrate lyase, a marker enzyme for the glyoxysomal matrix, was dissolved completely both in high-salt buffer and alkaline solution. In addition, Pex14p in intact glyoxysomes was sensitive to the digestion of proteinase K both in the absence and presence of Triton X-100, whereas isocitrate lyase was degraded only in the presence of Triton X-100 (Figure 4D). Overall results suggest that the 75 kDa protein is a peroxisomal membrane-associated protein, and that at least a part of the polypeptide is located in the cytosol. Intracellular transport of PTS1-containing proteins in the ped2 mutant To analyze the intracellular transport of PTS1-containing proteins in the ped2 mutant, we generated plants expressing a jellyfish green fluorescent protein (GFP)–PTS1 fusion protein (GFP–PTS1) in a ped2 background, as described previously (Mano et al., 1999). GFP–PTS1 consisted of GFP fused to a dodecapeptide containing serine-lysine-leucine at the C-terminal end. These plants were created by outcrossing the ped2 mutant with transgenic Arabidopsis expressing GFP–PTS1. Additional control plants were created by outcrossing the ped2 mutant with transgenic Arabidopsis expressing only GFP. When GFP–PTS1 is expressed in cells of the F3 progeny that are homozygous for the ped2 allele, green fluorescence was observed both in the periphery of the cells (Figure 5A, arrow) and in small spots distributed diffusely throughout the periphery (Figure 5A, arrowhead). The fluorescence detected in the periphery indicated that a part of the GFP–PTS1 remains in the cytosol, since GFP without PTS1 showed a similar fluorescent pattern in the ped2 mutant (Figure 5B, arrow) and in wild-type plants (data not shown). In Figure 5A and B, the dark space surrounded by the cytosol corresponds to a central vacuole. The fluorescent spots distributed in the cytosol indicate that a significant amount of GFP–PTS1 was recognized correctly and transported into the peroxisomes in the cells of the ped2 mutant. In contrast, only punctate fluorescence was observed when GFP–PTS1 was expressed in wild-type plants (Figure 5C). These data indicate that the ability for intracellular transport of PTS1-containing proteins is reduced in the ped2 mutant. Figure 5.Subcellular localization of GFP–PTS1 fusion protein in ped2 mutant. Seedlings were grown under continuous illumination for 10 days. Images of the green fluorescence derived from GFP in root cells were taken by a confocal laser microscope as single optical sections. (A) Subcellular localization of GFP–PTS1 expressed in the cells of a ped2 mutant. (B) Subcellular localization of GFP expressed in the cells of a ped2 mutant. (C) Subcellular localization of GFP–PTS1 expressed in the cells of a wild-type plant. (D) No fluorescence was observed in non-transformed cell of a ped2 mutant. Arrowheads indicate fluorescence detected in peroxisomes, whereas the arrows indicate fluorescence detected in the cytosol. Bar in (D), 20 μm. Magnifications of (A)–(D) are the same. Download figure Download PowerPoint Intracellular transport of PTS2-containing proteins in the ped2 mutant To analyze intracellular transport of PTS2-containing proteins in the ped2 mutant, two PTS2-containing proteins, 3-ketoacyl CoA thiolase (Figure 6A, ped2) and malate dehydrogenase (Figure 6B, ped2), were analyzed in 3- and 5-day-old etiolated cotyledons and 7-day-old green cotyledons by using an immunoblotting technique. As shown in Figure 6A, 3-day-old etiolated cotyledons contained two types of 3-ketoacyl CoA thiolase. One of these corresponds to the mature form of 3-ketoacyl CoA thiolase (45 kDa) (Figure 6A, arrowhead), whereas the other was an additional protein with a higher molecular mass (48 kDa) (Figure 6A, arrow). We have demonstrated previously that the larger protein corresponded to the precursor form of 3-ketoacyl CoA thiolase that accumulated in the cytosol (Kato et al., 1996a; Hayashi et al., 1998). In addition, 3-day-old etiolated cotyledons contained the mature form of malate dehydrogenase (33 kDa) (Figure 6B, arrowhead) and the precursor form of the enzyme (37 kDa) (Figure 6B, arrow). In contrast, the wild-type plants did not contain detectable amounts of the precursor proteins during any stages of post-germinative growth (Figure 6A and B, WT). The precursor proteins detected in 3-day-old etiolated cotyledons of the ped2 mutant rapidly disappeared, whereas the amounts of the mature proteins remained at similar levels during subsequent seedling growth. 3-Ketoacyl CoA thiolase and malate dehydrogenase are known to be actively synthesized in cells of etiolated cotyledons but not in the green cotyledons (Kato et al., 1996a, 1998). Accumulation of the precursors for PTS2-containing proteins in the ped2 mutant occurred only during the period of active protein synthesis. These data indicate that the ped2 mutant has reduced activity for the intracellular transport of PTS2-containing proteins, and is not able to import all of the PTS2-containing proteins when these proteins are actively synthesized. Figure 6.Immunodetection of thiolase and malate dehydrogenase in cotyledonary cells of ped2 mutants. Seedlings of the ped2 mutant (ped2) and wild-type plant (WT) were grown in continuous darkness for 3 days (3D), 5 days (5D) or under continuous illumination for 7 days (7L). Ten micrograms of total protein prepared from the cotyledons were subjected to immunoblotting using an antibody raised against 3-ketoacyl CoA thiolase (A) and malate dehydrogenase (B). Arrowheads indicate the positions of the mature proteins, whereas the arrows indicate the positions of the precursors. Download figure Download PowerPoint Morphology of glyoxysomes, leaf peroxisomes and unspecialized peroxisomes in the ped2 mutant Figure 7 shows an immunoelectron microscopic analysis of various peroxisomes in wild-type plants and the ped2 mutant. As mentioned above, there are three types of plant peroxisomes: glyoxysomes, leaf peroxisomes and non-specialized peroxisomes. In wild-type plants, these peroxisomes have similar morphologies (Nishimura et al., 1996). As shown in Figure 7A, glyoxysomes found in the 5-day-old etiolated cotyledons of wild-type plants are ∼0.5 μm in diameter and have a round or oval shape containing a uniform matrix. The glyoxysomes contain enzymes for fatty acid β-oxidation. When cells were immunogold labeled using antibodies raised against one of these enzymes, 3-ketoacyl CoA thiolase, the gold particles were exclusively localized on the glyoxysomes (Figure 7A). In contrast, the peroxisomes in the ped2 mutant showed an abnormal morphology (Figure 7B–E). Glyoxysomes found in the etiolated cotyledons of the ped2 mutant were shrunken and not round (Figure 7B). Therefore, they looked very different from the glyoxysomes of wild-type plants. A small but significant number of gold particles were detected when the glyoxysomes of the ped2 mutant were stained with antibodies raised against 3-ketoacyl CoA thiolase. In contrast, the number of gold particles detected in the cytosol was not significant, in spite of the fact that the precursor of 3-ketoacyl CoA thiolase is accumulated in the cytosol. This may be because the concentration of the precursor that accumulated in the cytosol was not sufficient to be clearly detected. A similar abnormal morphology was detected in leaf peroxisomes found in cells of green cotyledons (Figure 7C) and leaves (Figure 7D), as well as in unspecialized peroxisomes found in cells of root (Figure 7E). Since the leaf peroxisomes contain photorespiration enzymes, such as hydroxypyruvate reductase, the leaf peroxisomes were stained using antibodies raised against hydroxypyruvate reductase (Figure 7C and D). However, fewer gold particles were detected in the leaf peroxisomes of the ped2 mutant than in those of the wild-type plant (data not shown). A similar phenomenon was observed when unspecialized peroxisomes in root cells were stained with antibodies raised against catalase (Figure 7E). These data indicate that all three kinds of peroxisome in the ped2 mutant have abnormal morphologies, and contain fewer enzymes than do the peroxisomes of wild-type plants. Figure 7.Electron microscopic