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Richard N. Trelease - One of the best experts on this subject based on the ideXlab platform.
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Identification of Porin-Like Polypeptide(s) in the Boundary Membrane of Oilseed Glyoxysomes
Plant and Cell Physiology, 2000Co-Authors: Francisco J. Corpas, Michael J Brown, Luisa M Sandalio, Richard N. TreleaseAbstract:;A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling Glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling Glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaftype peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of Glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) Glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.
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diverse amino acid residues function within the type 1 peroxisomal targeting signal implications for the role of accessory residues upstream of the type 1 peroxisomal targeting signal
Plant Physiology, 1997Co-Authors: Robert T Mullen, M S Lee, Charles R Flynn, Richard N. TreleaseAbstract:The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to Glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into Glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to Glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting. The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to Glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into Glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to Glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting. The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to Glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into Glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to Glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting. The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to Glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into Glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to Glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting.
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Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to Glyoxysomes.
The Plant Cell, 1997Co-Authors: Robert T Mullen, Richard N. TreleaseAbstract:Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into Glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the Glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides were still acquired by Glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subunits are being piggybacked as multimers to Glyoxysomes by association with subunits possessing a PTS1. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.
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four putative glyoxysome membrane proteins are instead immunologically related protein body membrane proteins
Plant Science, 1995Co-Authors: Jeff Bunkelmann, Francisco J. Corpas, Richard N. TreleaseAbstract:Abstract Previous research revealed that peroxisomes/Glyoxysomes in plants, mammals, and yeasts possessed a prominent, integral peroxisome membrane protein (PMP) with a molecular mass in the range 21–26 kDa. We also found a major, low molecular mass polypeptide in the glyoxysome membrane fractions from four oilseed species: putative PMP26 in cotton (Gossypium hirsutum L.), PMP22 in cucumber (Cucumis sativus L.), PMP24.5 in sunflower (Helianthus annuus L.), and PMP24 in castor bean (Ricinus communis L.). Rabbit antiserum, produced against all the proteins which were solubilized from membranes recovered from isolated cotton glyoxysome fractions, recognized these four putative PMPs on Western blots as well as similar molecular mass polypeptides in mitochondria and protein bodies isolated from cotton and cucumber cotyledons. Postembedding, immunogold analyses of cotton and cucumber cotyledons, however, revealed that antibodies which were affinity-purified to the putative cotton glyoxysome 26-kDa polypeptide specifically bound to membranes of protein bodies, but not to membranes of Glyoxysomes nor mitochondria. Antibodies to the bean tonoplast intrinsic protein (α-TIP), a known protein body membrane protein (PBMP), also recognized the four putative PMPs on Western blots and co-localized with anti-cotton PMP26 IgGs on thin sections to membranes of protein bodies. Intact protein bodies were not the source of the PBMPs in the oilseed organelle fractions; immunogold labeling revealed immunoreactive, small indiscrete vesicles interspersed among Glyoxysomes and mitochondria in gradient fractions. Thus, putative prominent PMPs believed previously by us and others to be important in biogenesis and/or function of plant peroxisomes were discovered to be antigenically-related PBMPs of similar low molecular mass whose function in oilseeds has yet to be elucidated.
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Identification and immunochemical characterization of a family of peroxisome membrane proteins (PMPs) in oilseed Glyoxysomes.
European Journal of Cell Biology, 1994Co-Authors: Francisco J. Corpas, Bunkelmann J, Richard N. TreleaseAbstract:Prior to this study the only antibodies available for characterizing peroxisome membrane proteins (PMPs) in plants were the antibodies raised against membranes isolated from castor bean endosperm Glyoxysomes by Halpin et al. (Planta 179, 331-339 (1989)). We raised antibodies to four different nondenatured PMP complexes solubilized in 0.63 M aminocaproate/1% dodecylmaltoside from alkaline carbonate-washed, cucumber cotyledon glyoxysome membranes. The four complexes, approximately 290/270, 148, 128 and 67 kDa, were excised from 5 to 10% nondenaturing gradient gels, passively eluted from their homogenized gel slice, concentrated, then injected subcutaneously into rabbits. SDS-PAGE (10-15% gradient) of the total detergent-solubilized PMPs revealed six prominent membrane polypeptides: 73, 61, 52, 36, 30, and 22 kDa. The SDS-PMP composition of each nondenatured antigen was: PMP290/270-52, 30, 28 kDa; PMP148-30, 28, 26, 23, 22 kDa; PMP 128-73, 66, 36, 30, 23 kDa; PMP67-34, 30 kDa. These data indicated that several prominent as well as several minor polypeptides were common components of the PMP complexes. Three of the four antisera to the complexes were polyspecific, recognizing several of these common SDS polypeptides, whereas the fourth antiserum, anti-PMP67, was monospecific for PMP30. Cross-reactivities were evident with each antiserum to several of these SDS PMPs from castor bean, cotton and sunflower. Affinity-purified anti-PMP30 and anti-PMP73 antibodies specifically bound to the boundary membrane of cucumber Glyoxysomes in cells examined by indirect, postembedment (LR White), immunocytochemistry. These, and the family of other antibodies produced in this study, provide specific molecular probes essential for elucidating biogenesis and discovering function(s) of the integral membrane proteins in oilseed Glyoxysomes.
Alison Baker - One of the best experts on this subject based on the ideXlab platform.
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NADPH is a specific inhibitor of protein import into Glyoxysomes
Plant Journal, 1998Co-Authors: Martin R. Pool, Eduardo Lopez-huertas, Jim-tong Horng, Alison BakerAbstract:Summary We have studied the import of proteins into Glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into Glyoxysomes, and inhibition is determined by the ratio of NADP+ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of Glyoxysomes with NADPH, followed by re-isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP+; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as Glyoxysomes are converted to leaf-type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.
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Castor Bean Isocitrate Lyase Lacking the Putative Peroxisomal Targeting Signal 1 ARM Is Imported into Plant Peroxisomes Both in Vitro and in Vivo
Plant physiology, 1996Co-Authors: X Gao, Martin R. Pool, Joanne L. Marrison, R. M. Leech, Alison BakerAbstract:To understand and manipulate plant peroxisomal protein targeting, it is important to establish the universality or otherwise of targeting signals. Contradictory results have been published concerning the nature and location of the glyoxysomal/peroxisomal targeting signal of isocitrate lyase (ICL). L.J. Olsen, W.F. Ettinger, B. Damsz, K. Matsudaira, A. Webb, and J.J. Harada ([1993] Plant Cell 5: 941–952) concluded that the last 5 amino acids (AKSRM) of Brassica napus ICL were sufficient and the last 37 amino acids were necessary for targeting to Arabidopsis leaf peroxisomes. In contrast, R. Behari and A. Baker ([1993] J Biol Chem 268: 7315–7322) could find no requirement for the almost identical carboxy-terminal sequence AKARM for import of Ricinus communis ICL into isolated sunflower cotyledon Glyoxysomes. To resolve this discrepancy, the import characteristics of a mutant R. communis ICL lacking the last 19 amino acids of the carboxy terminus was studied. ICL[delta]19 was able to be imported by isolated sunflower Glyoxysomes and by tobacco leaf peroxisomes when expressed transgenically. These results demonstrate that the in vitro import system faithfully reflects targeting in vivo, and that the source of the organelles (Arabidopsis versus sunflower, leaf peroxisomes versus seed Glyoxysomes) is not responsible for observed differences between B. napus and R. communis ICL. The R. communis enzyme would therefore appear to possess an additional glyoxysome/peroxisome targeting signal that is lacking in the B. napus protein.
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Investigation of the energy requirement and targeting signal for the import of glycolate oxidase into Glyoxysomes.
FEBS Journal, 1995Co-Authors: Jim-tong Horng, Richa Behari, L. E. Carol‐ann Burke, Alison BakerAbstract:The uptake of glycolate oxidase into peroxisomes has been studied using an in vitro import system. Import of glycolate oxidase was found to be ATP-dependent and temperature-dependent and specific for Glyoxysomes. In these respects it resembles the import of isocitrate lyase into both Glyoxysomes and leaf-type peroxisomes; thus the ATP-dependence and temperature dependence appear to be general properties of plant microbody protein import. Two mutant versions of glycolate oxidase were prepared lacking 59 amino acids of the N-terminus and 53 amino acids of C-terminus, respectively. Both were capable of ATP-dependent import, whereas a fusion protein consisting of the cytosolic protein dihydrofolate reductase linked to the last 20 amino acids of glycolate oxidase bound to Glyoxysomes but did not enter the organelle.
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The carboxyl terminus of isocitrate lyase is not essential for import into Glyoxysomes in an in vitro system.
Journal of Biological Chemistry, 1993Co-Authors: Richa Behari, Alison BakerAbstract:Abstract A procedure has been established for the isolation of intact and import-competent Glyoxysomes from the cotyledons of sunflower (Helianthus annuus) seedlings. Radiolabeled isocitrate lyase (a glyoxysomal matrix protein) was produced by in vitro transcription of the castor bean cDNA and translation of the RNA in a wheat germ lysate. The heterologous isocitrate lyase was imported into isolated sunflower Glyoxysomes and protease-protected. Dihydrofolate reductase, a cytoplasmic protein, neither bound to nor imported into the Glyoxysomes. Import of isocitrate lyase was not observed when the glyoxysome fraction was replaced by a mitochondrial fraction. The import of isocitrate lyase into Glyoxysomes was temperature- and ATP-dependent. Progressive carboxyl-terminal truncations of the isocitrate lyase gene were transcribed and translated to yield polypeptides with the same amino terminus but lacking varying amounts of the carboxyl terminus. All these polypeptides imported with the same characteristics as the full-length protein, suggesting that targeting information must be present within the first 168 amino acids, and, unlike some other peroxisomal proteins, the carboxyl terminus is dispensable for targeting and import.
Mikio Nishimura - One of the best experts on this subject based on the ideXlab platform.
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Novel Glyoxysomal Protein Kinase, GPK1, Identified by Proteomic Analysis of Glyoxysomes in Etiolated Cotyledons of Arabidopsis thaliana
Plant and Cell Physiology, 2003Co-Authors: Yoichiro Fukao, Ikuko Hara-nishimura, Makoto Hayashi, Mikio NishimuraAbstract:Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of Glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in Glyoxysomes. To better understand the functions of Glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase 1 (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in Glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the Glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [γ- 3 2 P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immuno-inhibition of phosphorylation in Glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in Glyoxysomes.
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direct interaction between Glyoxysomes and lipid bodies in cotyledons of the arabidopsis thaliana ped1 mutant
Protoplasma, 2001Co-Authors: Y Hayashi, Makoto Hayashi, H Hayashi, Ikuko Haranishimura, Mikio NishimuraAbstract:During germination and subsequent growth of fatty seeds, higher plants obtain energy from the glyconeogenic pathway in which fatty acids are converted to succinate in Glyoxysomes, which contain enzymes for fatty acid beta-oxidation and the glyoxylate cycle. The Arabidopsis thaliana ped1 gene encodes a 3-ketoacyl-CoA thiolase (EC 2.3.1.16) involved in fatty acid beta-oxidation. The ped1 mutant shows normal germination and seedling growth under white light. However, etiolated cotyledons of the ped1 mutant grow poorly in the dark and have small cotyledons. To elucidate the mechanisms of lipid degradation during germination in the ped1 mutant, we examined the morphology of the ped1 mutant. The Glyoxysomes in etiolated cotyledons of the ped1 mutant appeared abnormal, having tubular structures that contained many vesicles. Electron microscopic analysis revealed that the tubular structures in Glyoxysomes are derived from invagination of the glyoxysomal membrane. By immunoelectron microscopic analysis, acyl-CoA synthetase (EC 6.2.1.3), which was located on the membrane of Glyoxysomes in wild-type plants, was located on the membranes of the tubular structures in the Glyoxysomes in the ped1 mutant. These invagination sites were always in contact with lipid bodies. The tubular structure had many vesicles containing substances with the same electron density as those in the lipid bodies. From these results, we propose a model in which there is a direct mechanism of transporting lipids from the lipid bodies to Glyoxysomes during fatty acid beta-oxidation.
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microbody defective mutants of arabidopsis
Journal of Plant Research, 1998Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Kanako Toriyama, Shoji Mano, Katsushi Yamaguchi, Maki Kondo, Hiroshi HayashiAbstract:In germinating fatty seedlings, microbodies are differentiated to leaf peroxisomes from Glyoxysomes during greening, and then transformed to Glyoxysomes from leaf peroxisomes during senescence. These transformations of microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described.
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transport of chimeric proteins that contain a carboxy terminal targeting signal into plant microbodies
Plant Journal, 1996Co-Authors: Makoto Hayashi, Akira Kato, Maki Kondo, Masahiro Aoki, Mikio NishimuraAbstract:Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser-Arg-Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of beta-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into Glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into Glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeptides with S, A or P at the -3 position, K or R at the -2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.
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Functional Transformation of Microbodies in Higher Plant Cells
Cell structure and function, 1996Co-Authors: Mikio Nishimura, Makoto Hayashi, Akira Kato, Katsushi Yamaguchi, Shoji ManoAbstract:In germinating fatty seedlings, microbodies are functionally transformed to leaf peroxisomes from Glyoxysomes during greening, and then converted to Glyoxysomes from leaf peroxisomes during senescence. Immunocytochemical studies revealed that Glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again directly converted to Glyoxysomes during senescence. The reversible transformations of microbodies are regulated at various levels, such as gene expression, splicing of the mRNA and degradation of microbody proteins. The regulatory mechanisms underlying this organelle differentiation are described.
Laura J. Olsen - One of the best experts on this subject based on the ideXlab platform.
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isolation of Glyoxysomes from pumpkin cotyledons
Current protocols in pharmacology, 2005Co-Authors: Nicola J Harrisonlowe, Laura J. OlsenAbstract:Peroxisomes are single-membrane-bound organelles found in virtually all eukaryotes. In plants, there are several classes of peroxisomes. Glyoxysomes are found in germinating seedlings and contain enzymes specific for the glyoxylate cycle, including isocitrate lyase and malate synthase. After seedlings become photosynthetic, leaf peroxisomes participate in reactions of the photorespiration pathway and contain characteristic enzymes such as glycolate oxidase and hydroxypyruvate reductase. As leaves begin to senesce, leaf peroxisomes are transformed back into Glyoxysomes. Root peroxisomes in the nodules of legumes, for example, sequester enzymes such as allantoinase and uricase, which contribute to nitrogen metabolism in these tissues. Thus, peroxisomes participate in many metabolic pathways and contain specific enzyme complements, depending on the tissue source. All peroxisomes contain catalase to degrade hydrogen peroxide and enzymes to accomplish β-oxidation of fatty acids. Glyoxysomes can be isolated from pumpkin cotyledons by standard differential centrifugation and density separation, as described in this article. Keywords: Glyoxysomes; peroxisomes; protein import
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protein transport into higher plant peroxisomes in vitro import assay provides evidence for receptor involvement
Plant Physiology, 1997Co-Authors: Donna Garvey Brickner, John J. Harada, Laura J. OlsenAbstract:Peroxisome biogenesis requires that proteins be transported from their site of synthesis in the cytoplasm to their final location in the peroxisome matrix or membrane. Glyoxysomes are a class of peroxisomes found primarily in germinating seedlings and are involved in mobilizing fatty acids via the glyoxylate cycle and the [beta]-oxidation pathway. We have used an in vitro assay to study the mechanism(s) of import of proteins into Glyoxysomes. Results from this assay indicate that the transport process is time- and temperature- dependent and is specific for peroxisomal proteins. Isocitrate lyase, a glyoxysomal protein, and the leaf-type peroxisomal enzyme glycolate oxidase (GLO) were transported into pumpkin (Cucurbita pepo) Glyoxysomes with no apparent differences in efficiency of import. Thus, this in vitro assay appears to be physiologically relevant and correlates well with expected in vivo conditions. Protein import was also energy-dependent and saturable. Nonradiolabeled GLO competed with radiolabeled, in vitro-synthesized GLO for components of the import machinery. Finally, pretreatment of the isolated Glyoxysomes with protease virtually abolished subsequent import of GLO. Taken together, these results indicate that a proteinaceous receptor is involved in the import of peroxisomal proteins.
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Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant. Plant Cell 5:941–952
1993Co-Authors: Laura J. Olsen, Kelly B Matsudaira, Mary Alice Webbib, John J. HaradaAbstract:Higher plants possess several classes of peroxisomes that are present at distinct developmental stages and serve different metabolic roles. To investigate the cellular processes that regulate developmental transitions of peroxisomal function, we analyzed the targeting of glyoxysomal proteins to leaf-type and root peroxisomes. We transferred genes encoding the glyoxysome-specific enzymes isocitrate lyase (IL) and malate synthase into Arabidopsis plants and showed, in cell fractionation and immunogold localization experiments, that the glyoxysomal proteins were imported into leaf-type and root peroxisomes. We next defined the sequences that target IL to peroxisomes and asked whether the same targeting determinant is recognized by different classes of the organelle. By localizing deletion and fusion derivatives of IL, we showed that the polypeptide’s carboxyl terminus is both necessary for its transport to peroxisomes and sufficient to redirect a passenger protein from the cytosol to both Glyoxysomes and leaf-type peroxisomes. Thus, glyoxysomal proteins are transported into several classes of peroxisomes using a common targeting determinant, suggesting that protein import does not play a regulatory role in determining a peroxisome’s function. Rather, the specific metabolic role of a peroxisome appears to be determined primarily by processes that regulate the synthesis and/or stability of its constituent proteins. These processes are specified by the differentiated state of the cells in which the organelles are found
Helmut Kindl - One of the best experts on this subject based on the ideXlab platform.
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the membrane bound dnaj protein located at the cytosolic site of Glyoxysomes specifically binds the cytosolic isoform 1 of hsp70 but not other hsp70 species
FEBS Journal, 2000Co-Authors: Jorg Diefenbach, Helmut KindlAbstract:DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and Glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase–Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of Glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.
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thiolase mrna translated in vitro yields a peptide with a putative n terminal presequence
Plant Molecular Biology, 1993Co-Authors: Regina Preisigmuller, Helmut KindlAbstract:Thiolase is part of the fatty acid oxidation machinery which in plants is located within Glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into Glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.