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ABSTRACT: It is well known that peroxisomal matrix proteins contain one of two targeting signals, PTS1 and PTS2. We comprehensively surveyed genes related to peroxisomal function and biogenesis in the entire Arabidopsis genome sequence. Here, we identified 256 gene candidates of PTS1- and PTS2-containing proteins and another 30 genes of non-PTS-containing proteins. Of these, only 29 proteins have been reported to be functionally characterized as peroxisomal proteins in higher plants. We extensively investigated expression profiles of genes described above in various organs of Arabidopsis: Statistical analyses of these expression profiles revealed that peroxisomal genes could be divided into five groups. One group showed ubiquitous expression in all organs examined, while the other four were classified as showing organ-specific expression in seedlings, cotyledons, roots and in both cotyledons and leaves. These data proposed more detailed description of differentiation of plant peroxisomes.
Plant and Cell Physiology 01/2004; 44(12):1275-89. · 4.70 Impact Factor
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ABSTRACT: Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.
Biochemical and Biophysical Research Communications 07/2003; 306(1):169-76. · 2.48 Impact Factor
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ABSTRACT: In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designated AtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). The AtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that the AtLACS6 is localized on glyoxysomal membranes. AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and beta-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid beta-oxidation activating the fatty acids.
Plant physiology 01/2003; 130(4):2019-26. · 6.53 Impact Factor
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ABSTRACT: Pumpkin (Cucurbita sp.) long-chain acyl-CoA oxidase (ACOX) (EC 1.3.3.6) was purified to homogeneity by hydrophobic interaction, hydroxyapatite, affinity, and anion exchange chromatographies. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 72-kDa subunits. The protein is exclusively localized in glyoxysomes. The enzyme catalyzes selectively the oxidation of CoA esters of fatty acids with 12–18 C atoms and exhibits highest activity with C-14 fatty acids, but no activity with isobutyryl-CoA and isovaleryl-CoA (branched chain) or glutaryl-CoA (dicarboxylic). The enzyme is strongly inhibited by high concentrations of palmitoyl-CoA and weakly inhibited by high concentration of myristoyl-CoA. It is also inhibited by Triton X-100 at concentrations above 0.018% (w/v) the critical micellar concentration. The consequences of the substrate inhibition for the evaluation of long-chain ACOX activity in plant tissues are discussed.
Physiologia Plantarum 01/2002; 106(2):170 - 176. · 3.11 Impact Factor
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ABSTRACT: 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.
Journal of Plant Research 04/1998; 111(2):329-332. · 1.75 Impact Factor
