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Plant Cell Physiol. 42(8): 835–841 (2001) JSPP © 2001 Developmental Analysis of a Putative ATP/ADP Carrier Protein Localized on Glyoxysomal Membranes During the Peroxisome Transition in Pumpkin Cotyledons Youichiro Fukao 1, 2, Yasuko Hayashi 3, Shoji Mano 1, Makoto Hayashi 1 and Mikio Nishimura 1, 2, 4 1 Departmnt of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan Department of Molecular Biomechanics, School of Life Science, Graduate University for Advanced Studies, Okazaki, 444-8585 Japan 3 Departmnt of Environmental Science, Faculty of Science, Niigata University, Ikarashi, Niigata, 950-2181 Japan 2 ; oxidation cycle and the glyoxylate cycle, and leaf peroxisomes contain enzymes of the photorespiration pathway that act in concert with enzymes in chloroplasts and mitochondria (Tolbert et al. 1968). Peroxisomes are transformed from glyoxysomes to leaf peroxisomes in greening cotyledons of fatty seedlings, and this transformation is controlled by light (Nishimura et al. 1993, Titus and Becker 1985). The reverse conversion from leaf peroxisomes to glyoxysomes also occurs in senescing cotyledons (De Bellis and Nishimura 1991). In contrast to the matrix enzymes, how membrane proteins change during the peroxisome transition is not well understood. To date only a few peroxisomal membrane proteins (PMPs) have been characterized in higher plants, for example PMP31 (peroxisomal ascorbate peroxidase), PMP28 and PMP22 (Yamaguchi et al. 1995a, Tugal et al. 1999). In this paper, we report the presence of a novel PMP, PMP38, in higher plant peroxisomes as an integral membrane protein. The deduced amino acid sequence of the cDNA of Arabidopsis thaliana has high similarity with those of Homo sapiens (Hs) PMP34 and Candida boidinii (Cb) PMP47. Developmental analysis revealed that the level of PMP38 was similar to those of the fatty acid >-oxidation enzymes during greening and senescence, suggesting that PMP38 functions in concert with the fatty acid >-oxidation cycle. In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid >-oxidation cycle, that are localized in the matrix of glyoxysomes. Key words: ATP/ADP carrier protein — Fatty acid >-oxidation cycle — Glyoxysome — Integral membrane protein — The peroxisome transition. Results Abbreviations: AAC, ATP/ADP carrier protein; mPTS, membrane peroxisomal targeting signal; PMP, peroxisomal membrane protein. Deduced amino acid sequences of AtPMP38 To identify the major PMPs, we prepared the pumpkin glyoxysomal membrane fraction and determined the internal amino acid sequences of some major membrane proteins by ingel digestion (data not shown). By this procedure, we obtained one internal amino acid sequence (IQELYDEAGI) from the polypeptides with a molecular mass of about 38 kDa, designated PMP38 from the molecular mass. This sequence had high homology with the Arabidopsis putative peroxisomal ATP/ADP carrier protein (AAC) using the BLAST algorithm (Altschul et al. 1990). To characterize this protein, we obtained EST clone (173G9T7) corresponding to the sequences of putative peroxisomal AAC and determined the sequences. As shown in Fig. 1, the complete sequence of the cDNA of A. Introduction The peroxisome is a single membrane-bound organelle present in eukaryotic cells (Lazarow and Fujiki 1985). In higher plants, peroxisomes are classified into three functionally different types, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes (Beevers 1979, Huang et al. 1983). Enzymatic components of peroxisomes have been characterized well. Glyoxysomes contain enzymes for the fatty acid >4 Corresponding author: E-mail, [email protected]; Fax, +81-564-55-7505. 835 836 Glyoxysomal ATP/ADP carrier protein Fig. 2 Comparison of energy-transfer-protein signatures of AtPMP38, HsPMP34 and CbPMP47. Energy-transfer-protein signature of CbPMP47 is single underlined and transmembrane domains are double underlined according to the transmembrane domain of AtPMP38; white letters indicate the corresponding conserved amino acid residue. The alignment only shows the conserved energy-transferprotein signature. The sequence alignment was performed using the CLUSTAL W program. Fig. 1 Comparison of the deduced amino acid sequences with AtPMP38 and HsPMP34 and CbPMP47. (A) The internal amino acid sequence corresponding to the pumpkin mature protein, as determined by Edman degradation, is single underlined, the putative mPTS motif is double underlined and putative transmembrane domains are indicated by dotted underlines; white letters indicate corresponding conserved amino acid residue. The sequence alignment was performed using the CLUSTAL W program. (B) Hydropathy plots of AtPMP38, HsPMP34 and CbPMP47. Hydrophobic residues are depicted as positive and hydrophilic residues as negative. The putative transmembrane domains are indicated by bars. At, Arabidopsis thaliana; Hs, Homo sapience; Cb, Candida boidinii. thaliana revealed an open reading frame encoding for a 331amino acid protein with a calculated molecular mass of 36,171 Da. The alignment of peroxisomal AAC homologues of other organisms showed 25% identity to the HsPMP34 proteins, and 26% identity to the CbPMP47 proteins (Wylin et al. 1998, McCammon et al. 1990). However, although AtPMP38 showed low similarity (14% identity) to the putative mitochondrial AAC of A. thaliana that had no membrane peroxisomal targeting signal (mPTS), AtPMP38 has the energy-transfer- protein signature that is a conserved residue characteristic of peroxisomal AAC homologues of other organisms, such as HsPMP34 and CbPMP47, and mitochondrial AAC homologues of Saccharomyces cerevisiae that is located predominantly at the borders of the transmembrane domains as shown in Fig. 2 (Bairoch 1993). The hydropathy plots of AtPMP38, HsPMP34 and CbPMP47 (analyzed with the algorithm of von Heijne) show that these proteins belong to the six-transmembrane family of transport systems (Fig. 1), supporting that PMP38 is a member of the AAC family. AtPMP38 has a putative mPTS between putative transmembrane domains 4 and 5 (Fig 1A). This motif was determined on CbPMP47 sequences (Dyer et al. 1996). From this result, AtPMP38 was expected to be localized on peroxisomal membranes. We found this motif on other AtPMPs, although it is unknown yet whether the motif of AtPMP38 actually functions as mPTS. The genomic DNA sample digested with the restricted enzymes, which had no site within the cDNA, was hybridized to the AtPMP38 probe and gave one fragment. By contrast, the genomic DNA digested with restricted enzymes, which had a single site within the cDNA, gave two fragments (data not shown). The results of Southern analysis of A. thaliana genomic DNA probed with AtPMP38 suggested that AtPMP38 is a single-copy gene. The gene encoding AtPMP38 is located on chromosome II within BAC T28M21 (Gen Bank accession no. AAB95282). Assessment of antibody raised against AtPMP38 As shown in Fig. 3, the antibody raised against AtPMP38 cross-reacted with only a single polypeptide of about 38 kDa in the total homogenates from etiolated A. thaliana and pumpkin Glyoxysomal ATP/ADP carrier protein Fig. 3 Immuno-cross reactivity with antibody raised against AtPMP38. Homogenates of etiolated cotyledons of Arabidopsis thaliana (lane 1), of pumpkin (lane 2) and peroxisomal membrane fraction from etiolated cotyledons of pumpkin (lane 3) were applied to SDS-PAGE and subsequent immunoblotting. Molecular markers are indicated in kDa on the right. Fig. 4 Subcellular localization of PMP38 in etiolated pumpkin cotyledons. Extracts prepared from 5 d etiolated pumpkin cotyledons were fractionated after sucrose density gradient centrifugation. (A) Immunological detection of PMP38. Each fraction (50 and 5 ml) was analyzed by immunoblotting using antibodies raised against AtPMP38 and isocitrate lyase, respectively. (B) The distribution of COX and catalase was monitored by the determination of enzyme activity (COX, closed triangle and catalase, closed circle). Open circle, sucrose concentration (w/w). ICL, isocitrate lyase; COX, cytochrome c oxidase. 837 Fig. 5 Immunoelectron microscopic analysis of an etiolated cotyledon of Arabidopsis thaliana with an antibody raised against the AtPMP38. Arrowheads mark the gold particles. G, glyoxysome; O, oil body. Bar = 500 nm. cotyledons, and in the isolated peroxisomes, respectively, indicating that the antibody was monospecific. We concluded that the antibody cross-reacted with not only AtPMP38 but also PmPMP38, although it was indicated that the molecular mass of PmPMP38 was slightly higher than that of AtPMP38. Subcellular and suborganellar localizations of PMP38 To investigate the subcellular localization of the PMP38, homogenates from 5 d etiolated pumpkin cotyledons were subjected to sucrose density gradient centrifugation. Fractions obtained were analyzed using an immunoblot technique with antibodies raised against the AtPMP38 and isocitrate lyase. Isocitrate lyase was used as a marker for glyoxysomal enzymes. As shown in Fig. 4A, PMP38 and isocitrate lyase were present together in fractions 23–25, as well as catalase activities (Fig. 4B). By contrast, no PMP38 was detected in fractions 11–15, which corresponds to the activity of a mitochondrial marker enzyme, cytochrome c oxidase. These results indicated that PMP38 is localized in glyoxysomes, but not in mitochondria. Fig. 5 shows an immunoelectron microscopic analysis of PMP38 in the cells of 5 d etiolated cotyledons of A. thaliana. The gold particles were detected in glyoxysomes, especially on glyoxysomal membranes. The particles were not detectable in any other organelles such as oil bodies. These results clearly indicated that the PMP38 is exclusively localized on glyoxysomal membranes. To investigate the localization of the PMP38 in glyoxysomes, we subfractionated glyoxysomes of pumpkin cotyledons into four fractions as reported previously (Yamaguchi et al. 1995b). Most matrix proteins were recovered in the water(lane 2) and salt-soluble (lane 3) fractions as shown for catalase (Fig. 6D). As shown in Fig. 6B, PMP38 was found in the alkali-insoluble fraction (lane 5). PMP38 was hardly extracted from glyoxysomes even with alkali solution. However, small amounts of thiolase (a matrix protein) were found in the alkali- 838 Glyoxysomal ATP/ADP carrier protein Fig. 6 SDS-PAGE and immunoblot analysis of AtPMP38 (B), peroxisomal ascorbate peroxidase (C) and thiolase (D). The various solubilized fractions of glyoxysomal membrane proteins were prepared using pumpkin cotyledons as described in “Materials and Methods” and subjected to SDS-PAGE, subsequent staining with Coomassie Brilliant Blue (A) and immunoblotting (B, C and D). Lane 1, isolated glyoxysomes; lane 2, the water-soluble fraction; lane 3, the salt-soluble fraction; lane 4, the alkali-soluble fraction and lane 5, the alkaliinsoluble fraction. Black arrow indicates the bands of AtPMP38. White and gray arrows indicate the bands of thiolase and peroxisomal ascorbate peroxidase, respectively. Molecular markers are indicated in kDa on the right. soluble and insoluble fractions (lane 4 and 5), indicating that both fractions were slightly contaminated with matrix proteins. Peroxisomal ascorbate peroxidase, another peroxisomal membrane protein, was mainly detected in the alkali-soluble fraction (lane 4). These results indicated that PMP38 is a glyoxysomal integral membrane protein. This is also supported by the hydropathy plot that shows six-transmembrane domains (Fig. 1B). Developmental changes in the level of PMP38 Fig. 7 shows the changes in the level of PMP38 during the post-germinative growth of the pumpkin seedlings. An immunoblot analysis of the cotyledons of the pumpkin seedlings grown in the dark showed that PMP38, as well as peroxisomal ascorbate peroxidase, glyoxysomal membrane proteins, and short-chain acyl-CoA oxidase and isocitrate lyase, glyoxysomal matrix proteins, reached a maximum level 6 d after germination. These enzymes were still present in the etiolated cotyledons 9 d after germination, although their levels were gradually decreased. After illumination of the seedlings, the amount of these enzymes was markedly decreased, but PMP38 was still present in greening cotyledons in a low amount like another enzyme of the fatty acid >-oxidation cycle, the short-chain acyl-CoA oxidase, and a related enzyme, peroxisomal ascor- Fig. 7 Developmental changes in the level of proteins for PMP38. Total homogenate prepared from pumpkin cotyledons (10–20 mg) was subjected to SDS-PAGE, which was followed by immunoblotting with antibodies raised against two peroxisomal membrane proteins, AtPMP38 and peroxisomal ascorbate peroxidase (pAPX), two glyoxysomal enzymes, short-chain acyl-CoA oxidase (SACOX) and isocitrate lyase (ICL) and leaf-peroxisomal enzymes, hydroxypyruvate reductase (HPR). Fig. 8 Changes in levels of peroxisomal enzymes during senescence in vitro. Green pumpkin cotyledons grown in a greenhouse for 20 d were placed in a plastic case in darkness. Total protein from cotyledons kept in darkness for 0, 2, 4, 6 and 8 d was subjected to SDSPAGE (10 mg each), which was followed by immunoblotting with antibodies raised against two peroxisomal membrane proteins, AtPMP38 and peroxisomal ascorbate peroxidase (pAPX), two glyoxysomal enzymes, short-chain acyl-CoA oxidase (SACOX) and isocitrate lyase (ICL) and leaf-peroxisomal enzymes, hydroxypyruvate reductase (HPR). Glyoxysomal ATP/ADP carrier protein bate peroxidase. These suggest that PMP38 is a glyoxysomal protein and may function in the fatty acid >-oxidation cycle. While hydroxypyruvate reductase, a matrix protein of leaf peroxisomes, was slightly detectable in pumpkin seedlings grown in the dark, it was increased markedly after illumination of the seedlings. We also investigated the changes in the levels of PMP38 during in vivo senescence. A small amount of PMP38 protein was present in cotyledons after 20 d in continuous light (0 d of senescence), and not changed during further incubation in continuous darkness (2–8 d) (Fig. 8). The developmental pattern was similar to those of peroxisomal ascorbate peroxidase and short-chain acyl-CoA oxidase, whereas the amount of isocitrate lyase increased markedly and the amount of hydroxypyruvate reductase gradually decreased during senescence. These results indicated that PMP38 plays a role during senescence as well as germination, and may act in concert with short-chain acyl-CoA oxidase and peroxisomal ascorbate peroxidase. Discussion PMP38 is a candidate of ATP/ADP carrier protein on glyoxysomal membranes AAC has been studied well in mitochondria and plastids. Generally, the mitochondrial AAC exchanges ATP synthesized in the matrix with ADP in the cytosol (Klingenberg 1989). Since ATP-synthesis pathways have not been found in the peroxisomes, PMP38 seems to import ATP into peroxisomes. PMP38 may play a role in glyoxysomal specific events, because the developmental pattern is similar to those of glyoxysomal enzymes (Fig. 7). Two metabolic pathways are known in glyoxysomes, namely glyoxylate cycle and fatty acid >oxidation cycle. The proteins of the fatty acid >-oxidation enzymes, such as short-chain acyl-CoA oxidase, increase during the early stage of germination in continuous darkness, reached the maximum level at 5–6 d like the developmental pattern of PMP38, and then decrease after exposure to light. After the completion of the peroxisome transition, proteins of the fatty acid >-oxidation enzymes were still detectable in leaf peroxisomes like the developmental pattern of PMP38, whereas the proteins of glyoxylate cycle, such as isocitrate lyase, disappeared (Fig. 7). They showed a similar pattern during senescence (Fig. 8). In fact, it was reported recently that the PMP47-deleted strain of C. boidinii could not grow well on a middle-chain fatty acid, laureate, but showed growth similar to that of the wild-type strain when provided with lauroyl-CoA (Nakagawa et al. 2000), suggesting that CbPMP47 is involved in the transport of ATP required in the conversion of laureate to lauroyl-CoA in peroxisomes. Altogether, we conclude that PMP38 may be a transporter involved in the transport of ATP that is required for the reaction of fatty acyl-CoA synthesis and functions in concert with enzymes participating in the fatty acid >-oxidation cycle. We are now examining the activity of the expressed PMP38 as an 839 ATP/ADP carrier. We have been screening for PMP38 knockout mutants of A. thaliana and generating anti-sense transgenic plants to clarify whether PMP38 is responsible for the fatty acid >-oxidation cycle or not. Recently, we screened for ped mutants with defects in the fatty acid >-oxidation cycle (Hayashi et al. 1998, Hayashi et al. 2000). Since 2,4dichlorophenoxybutyric acid (2,4-DB) is metabolized to produce a herbicide, 2,4-D, by the action of the fatty acid >oxidation cycle in higher plants, wild-type plants cannot germinate in the presence of 2,4-DB (Wain and Wightman 1954). However, ped mutants could germinate in the presence of 2,4DB, because these mutants are defective in the fatty acid >oxidation cycle. Therefore, if PMP38 is involved in the fatty acid >-oxidation cycle, a mutant of PMP38 will be able to germinate on a 2,4-DB plate. Further analysis of these mutants will help us to clarify the functions of PMP38. Change in levels of PMP38 during the peroxisome transition The peroxisomal matrix enzymes can be classified into three categories; (1) the glyoxysome-specific enzymes such as malate synthase and citrate synthase, which are members of the glyoxylate cycle; (2) the leaf peroxisome-specific enzymes such as glycolate oxidase and hydroxypyruvate reductase, which function in glycolate metabolism of photorespiration; and (3) the enzymes present in glyoxysomes and leaf peroxisomes such as catalase and the enzymes of the fatty acid >oxidation cycle. In dark-grown cotyledons, the glyoxysomespecific enzymes increased with the germination and decreased gradually after reaching the maximum level. By contrast, the leaf peroxisome-specific enzymes were at a low level in darkgrown cotyledons. When the seedlings were illuminated, the glyoxysome-specific enzymes decreased rapidly whereas the leaf peroxisome-specific enzymes increased markedly. The developmental changes of the enzymes present in both peroxisomes (glyoxysome and leaf peroxisome) were similar to those of glyoxysome-specific enzymes. After exposure to light, the glyoxysome-specific enzymes disappeared, whereas enzymes present in both peroxisomes decreased but were still retained at a low level after completion of the peroxisome transition from glyoxysomes to leaf peroxisomes. In this report, we showed that a membrane protein, PMP38 increases in the dark and is retained at a low level during greening of pumpkin cotyledons, indicating that PMP38 belongs to the enzymes present in both peroxisomes like the enzyme of the fatty acid >-oxidation cycle. We have previously shown that the membrane proteins of glyoxysomes, peroxisomal ascorbate peroxidase (PMP31) and PMP28, undergo drastic changes during the peroxisome transition and PMP28 disappeared after completion of the peroxisome transition whereas peroxisomal ascorbate peroxidase remained at a low level like PMP38 (Yamaguchi et al. 1995b). These results clearly show that membrane proteins of glyoxysomes are degraded to a considerable extent and may be replaced by leaf peroxisomal membrane proteins during the 840 Glyoxysomal ATP/ADP carrier protein transition as the matrix enzymes of glyoxysomes. Further characterization of leaf peroxisomal membrane proteins will provide us information how these membrane proteins are exchanged during the transformation of glyoxysomes to leaf peroxisomes. Materials and Methods Plant materials Pumpkin (Cucurbita sp. cv. Kurokawa Amakuri) seeds were soaked overnight, planted in moist rock fiber (66R; Nitto Bouseki, Chiba, Japan) and allowed to germinate at 25°C in darkness. The etiolated cotyledons obtained from seedlings kept in darkness for 5 d after germination were used for the preparation of glyoxysomes and those kept in darkness for 1–9 d for the developmental analysis. Some seedlings were transferred to light after 5 d in darkness and the development of the green cotyledons was also analyzed. Seeds of A. thaliana (L.) Heynh. Ecotype Columbia were germinated at 22°C in darkness. Preparation of glyoxysomes Glyoxysomes were isolated from etiolated pumpkin cotyledons. Etiolated cotyledons (50 g FW) were harvested from pumpkin seedlings kept in darkness for 5 d after germination, and homogenized with 200 ml of grinding medium that contained 20 mM pyrophosphateHCl, pH 7.5, 1 mM EDTA and 0.3 M mannitol in a chilled Waring blender for 3 s twice. The homogenate was squeezed through four layers of cheesecloth. The residue was homogenized similarly with another 200 ml of grinding medium. The filtrates were combined and were centrifuged at 1,500´g for 10 min to remove plastids and cell debris. After centrifugation at 10,000´g for 20 min, the pellet was resuspended in 150 ml of grinding medium, and was centrifuged similarly. The resultant pellet was then resuspended in 5 ml of buffer A (10 mM HEPES-KOH, pH 7.2, 1 mM EDTA and 0.3 M mannitol) and was subjected to centrifugation in Percoll. A 4 ml aliquot of the suspension was layered directly on top of 30 ml of a 28% (v/v) solution of Percoll in 10 mM HEPES-KOH, pH 7.2, 1 mM EDTA and 0.3 M raffinose and was centrifuged at 40,000´g for 30 min with slow acceleration and deceleration. Glyoxysomes sedimented near the bottom of the self-generated gradient were collected with a Pasteur pipette after centrifugation at 5,000´g for 10 min in 4 volumes of buffer A. The pellet was suspended carefully in 1 ml of buffer A and was used as purified glyoxysomes for subsequent analysis. All procedures were carried out at 4°C. Characterization of glyoxysomal membrane proteins Purified glyoxysomes were pelleted again by centrifugation at 5,000´g for 10 min and were suspended in 200 ml of 10 mM HEPESKOH buffer, pH 7.2. After centrifugation at 100,000´g for 30 min, the supernatant was analyzed as the water-soluble fraction. The pellet was resuspended in 10 mM HEPES-KOH, pH 7.2, which contained 0.2 M KCl and was centrifuged again at 100,000´g for 30 min to yield a saltsoluble fraction. The pellet was extracted with 0.1 M Na2CO3 by means of a sodium carbonate procedure that removed peripheral membrane proteins (Fujiki et al. 1982). After centrifugation at 100,000´g for 30 min, the supernatant was neutralized by the addition of 1 M HCl and was analyzed as the alkali-soluble fraction. The pellet was resuspended in 100 mM HEPES-KOH, pH 7.2, and was used as the alkaliinsoluble fraction. All procedures were carried out at 4°C. Determination of N-terminal amino acid sequences of internal peptides of PmPMP38 SDS-PAGE (12.5% polyacrylamide) was performed by the method of Laemmli (1970) for the separation of glyoxysomal membrane proteins. The separated proteins were then stained with Coomassie Brilliant Blue. The bands corresponding to PmPMP38 were cut out from the blot, and incubated in 300 ml of a solution containing ammonium bicarbonate (pH 8.0) and 50% acetonitrile with shaking for 30 min twice. The supernatant was discarded. The gel was dried and was then incubated in 10 ml of 0.2 M ammonium bicarbonate (pH 8.0) for 5 min. The sample was digested with 10 ml of 0.1 mg ml–1 trypsin solution (Wako, Osaka, Japan) in 20 ml of 0.2 M ammonium bicarbonate (pH 8.0) for 16 h at 37°C. The reaction was stopped by the addition of 4 ml of trifluoroacetic acid (TFA). The peptides were collected by shaking in 300 ml of 0.1% TFA and 60% acetonitrile for 30 min twice and subjected to SMART system (Pharmacia, Uppsala, Sweden) to separate the peptides. The separated peptides were subjected to automatic Edman degradation on a peptide sequencer (model 473A; Applied Biosystems Inc., Foster City, CA, U.S.A.). Sequencing of AtPMP38 EST clones AtPMP38 EST clone (stock number 173G9T7) was obtained from ABRC. These were derived from a lZip-Lox plasmid (GIBCO BRL, MD, U.S.A.). The nucleotide sequences with the longest cDNA insert, designated 173G9T7 were determined with an automatic DNA sequencer (model 310; Perkin Elmer/Applied Biosystems) according to the manufacturer’s instructions. The nucleotide and the deduced amino acid sequences were analyzed using the Gene-Works program (Intelli Genetics, Mountain View, CA, U.S.A.). Preparation of a specific antibody raised against AtPMP38 The DNA fragment of AtPMP38, corresponding to amino acid residues 26–108, was inserted into pET32a vector (Novagen, Madison, WI, U.S.A.). A fusion protein between the N-terminal region of AtPMP38 and histidine tag was expressed in E. coli cells and was purified by column chromatography on Ni2+-resin. The purified protein in 1 ml of sterilized water was emulsified with an equal volume of Freund’s complete adjuvant (DIFCO, Detroit, MI, U.S.A.). The emulsion was injected (about 0.4 mg of protein) on the back of a rabbit and then was similarly given to the first injection. Blood was taken from an auricular vein 7 d after the second booster injection. The serum was used for immunoblotting and immunogold analysis. Subcellular fractionation Etiolated cotyledons of pumpkin 5 d after germination in darkness were homogenized in a Petri dish by chopping with a razor blade for 5 min in 10 ml of a medium that contained 150 mM Tricine-KOH (pH 7.5), 1 mM EDTA and 0.5 M sucrose. The homogenate was passed through four layers of cheesecloth. The filtrate (3 ml) was layered onto a sucrose gradient that contained a 1 ml cushion of 60% (w/w) sucrose and an 11 ml linear sucrose gradient from 30 to 60% without buffer. The gradient was centrifuged at 25,000´g for 3 h using a SW 28-2 rotor in an ultracentrifuge (XL-90; Beckman, Fullerton, CA, U.S.A.). After centrifugation, fractions (0.5 ml each) were collected with an automatic liquid fractionator (ALC-2L; Advantec, Tokyo, Japan). All procedures were carried out at 4°C. Ultrastructural analysis For immunoelectron microscopy, etiolated cotyledons of A. thaliana 5 d after germination in darkness were frozen with a high pressure freezing machine (model HPM 010, Bal-Tec Inc., Balzers, FL, U.S.A.) and the ice in the cells was substituted with acetone for 2 d at –85°C. After being washed with acetone, cotyledons were embedded in LR white resin. Sections were treated successively with 1% bovine serum albumin (BSA) for 30 min and antibody raised against PMP38 rabbit IgG (1 : 100) for 10 h at 4°C. After being washed with phos- Glyoxysomal ATP/ADP carrier protein phate-buffered saline, the sections were treated with colloidal gold conjugated protein A for 1 h at room temperature. After being washed with 0.1% BSA-C (Aurion, Netherlands) and distilled water, sections were stained with an aqueous solution of uranyl acetate and with lead citrate. All sections were examined with a transmission electron microscope (model 1200EX; JEOL, Tokyo, Japan). Immunoblot analysis Pumpkin cotyledons kept under various conditions were homogenized with extraction buffer [20 mM Tris-HCl (pH 6.8), 2% SDS, 10% b-mercaptoethanol, 24% glycerol and a protein inhibitor cocktail (Boehringer Mannheim)] and then the homogenates were heated at 95°C for 5 min. The samples were centrifuged at 15,000´g for 5 min, and the protein in the supernatant was quantified using a protein assay kit (Nippon Bio-Rad Laboratories, Tokyo, Japan) with bovine gamma albumin as a standard. The samples were subjected to SDS-PAGE on a 12.5% polyacrylamide gel as described by Laemmli (1970). Then, an immunoblot analysis was performed by the method of Towbin et al. (1979). Immunologic reactions were detected by monitoring the activity of horseradish peroxidase (ECL system; Amersham). The antibody raised against isocitrate lyase, peroxisomal ascorbate peroxidase, thiolase, short-chain acyl-CoA oxidase and hydroxypyruvate reductase were prepared as described previously (Maeshima et al. 1988, Nito et al. 2001, Kato et al. 1996, Hayashi et al. 1999). Acknowledgements We are grateful to M. Maeshima (Nagoya University) for kind donation of the antibody raised against isocitrate lyase from castor bean and N.-H. Chua (Rockefeller University) for the antibody raised against hydroxypyruvate reductase from spinach. This work was supported in part by a grant from the Research for the Future Program of the Japanese Society for the Promotion of Science (JSPSRFTF96L00407), and by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (12440231). 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