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Plant Physiology Jan 1998All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay...
All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.
Topics: Adenosine Triphosphate; Alcohol Oxidoreductases; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations, Divalent; Cations, Monovalent; Guanosine Triphosphate; Ionophores; Kinetics; Magnesium; Microbodies; Nigericin; Nucleotides; Plants; Valinomycin
PubMed: 9449847
DOI: 10.1104/pp.116.1.309 -
The Plant Journal : For Cell and... Nov 2009PEX7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing proteins. In the present study, using a green fluorescent protein...
PEX7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing proteins. In the present study, using a green fluorescent protein (GFP) fusion protein of PEX7 (GFP-PEX7), we analyzed the molecular function and subcellular localization of PEX7 in Arabidopsis thaliana. The overexpression of GFP-PEX7 resulted in defective glyoxysomal fatty acid beta-oxidation, but had no significant effect on leaf peroxisomal function. Analysis of the subcellular localization of GFP-PEX7 in transgenic Arabidopsis showed that GFP-PEX7 localizes primarily to the peroxisome. Transient expression of a C- or N-terminal fusion protein of PEX7 and yellow fluorescent protein (YFP) (PEX7-YFP and YFP-PEX7, respectively) in leek epidermal cells, using the particle bombardment technique, confirmed that fluorescent protein-tagged PEX7 localizes to peroxisomes in Arabidopsis. Immunoblot analysis revealed that GFP-PEX7 accumulates primarily in peroxisomal membrane fractions, whereas endogenous PEX7 was distributed evenly in cytosolic and peroxisomal membrane fractions, which indicated that both endogenous PEX7 and GFP-PEX7 are properly targeted to peroxisomal membranes. The results of bimolecular fluorescence complementation (BiFC) and yeast two-hybrid analyses showed that PEX7 binds directly to PTS2-containing proteins and PEX12 in the peroxisomal membrane. We used red fluorescent protein (tdTomato) fusion protein of PEX7 (tdTomato-PEX7) in several Arabidopsis pex mutants to identify proteins required for the targeting of PEX7 to peroxisomes in planta. The results demonstrated that pex14, pex13 and pex12 mutations disrupt the proper targeting of PEX7 to peroxisomes. Overall, our results suggest that the targeting of PEX7 to peroxisomes requires four proteins: a PTS2-containing protein, PEX14, PEX13 and PEX12.
Topics: Arabidopsis; Arabidopsis Proteins; Genes, Reporter; Peroxisomal Targeting Signal 2 Receptor; Peroxisomes; Protein Binding; Receptors, Cytoplasmic and Nuclear
PubMed: 19594707
DOI: 10.1111/j.1365-313X.2009.03970.x -
European Journal of Biochemistry Jan 1994Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described...
Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10,000 x g, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53-kDa protein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved as a membrane-bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabelled mevalonic acid to cotyledons and isolation of glyoxysomes yielded labelled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient.
Topics: Cell Fractionation; Centrifugation, Density Gradient; Endopeptidases; Gene Library; HSP40 Heat-Shock Proteins; Heat-Shock Proteins; Hot Temperature; Intracellular Membranes; Membrane Proteins; Mevalonic Acid; Molecular Weight; Organelles; Recombinant Proteins; Seeds; Subcellular Fractions; Tritium; Vegetables
PubMed: 8307022
DOI: 10.1111/j.1432-1033.1994.tb19914.x -
Plant Physiology Jul 2009Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy...
Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs.
Topics: Alternative Splicing; Amino Acid Sequence; Biological Evolution; Bryopsida; Chromatography; Cytoplasmic Structures; Endoplasmic Reticulum; Glyoxysomes; Molecular Sequence Data; Photosynthesis; Phylogeny; Plant Proteins; Sequence Alignment; Spores
PubMed: 19420327
DOI: 10.1104/pp.109.138123 -
The Plant Journal : For Cell and... Jul 1998Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme...
Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.
Topics: Amino Acid Sequence; Cloning, Molecular; Cotyledon; Escherichia coli; Evolution, Molecular; Genetic Complementation Test; Isoenzymes; Kinetics; Malate Dehydrogenase; Medicago sativa; Meristem; Molecular Sequence Data; Phylogeny; Plant Leaves; Plant Roots; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 9721676
DOI: 10.1046/j.1365-313x.1998.00192.x -
Plant Physiology Aug 1987The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and...
The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and imbibition, then increases again following germination. The research reported herein is a comparative study of the synthesis and acquisition of MS into glyoxysomes as they occur in maturing and germinated seeds. Rate-zonal centrifugation of cotyledon extracts revealed that the 5 Svedberg unit (S) cytosolic form of MS was the only form present at 42 days postanthesis (DPA) when activity was first detectable. At later stages (48 DPA, 0 day, 26 hours, and 48 hours), both the 5S and glyoxysomal 20S forms were present, with the 20S form becoming much more prevalent. Western blot analyses revealed that no other form(s) of MS were present in the phosphate-buffered gradients, and that 5S and 20S forms had the same subunit molecular weight in maturing and germinated seeds. Comparisons of radiospecific activity of MS immunoprecipitates following in vivo labeling with [(35)S]methionine for varying time intervals provided strong evidence for a 5S-precursor to 20S-product relationship during both seed maturation and seedling growth. Comparisons of MS labeled in vivo and in vitro in wheat germ and rabbit reticulocyte lysates programmed with poly(A)(+)RNA (from maturing and germinated seeds) revealed no detectable differences in subunit molecular weights. These results reinforced our other data indicating that MS was synthesized in the cytosol and acquired by glyoxysomes in both maturing and germinated cotton seeds without involvement of an intervening aggregate pool in the endoplasmic reticulum, or via processing of a cleavable precursor molecule. MS was translated from poly(A)(+)RNA extracted from 28 DPA cotton seeds. This was nearly 2 weeks before MS activity or protein was detected in vivo. This finding invites further study on the regulation of RNA transcripts during maturation.
PubMed: 16665609
DOI: 10.1104/pp.84.4.1350 -
Plant Physiology Apr 1997Peroxisome biogenesis requires that proteins be transported from their site of synthesis in the cytoplasm to their final location in the peroxisome matrix or membrane....
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.
Topics: Adenosine Triphosphate; Alcohol Oxidoreductases; Endopeptidases; Kinetics; Microbodies; Organelles; Plant Leaves; Plants; Receptors, Cytoplasmic and Nuclear; Vegetables
PubMed: 9112774
DOI: 10.1104/pp.113.4.1213 -
Molecules and Cells Jun 1998Two maize glyoxysomal genes expressed during germination, malate synthase (MS) and isocitrate lyase (ICL), were used to characterize the regulatory roles of the...
Two maize glyoxysomal genes expressed during germination, malate synthase (MS) and isocitrate lyase (ICL), were used to characterize the regulatory roles of the Viviparous-1 (Vp1) regulatory gene and abscisic aicd (ABA) in the induction of embryo quiescence during kernel development. In wild-type maize embryo, MS and ICL transcripts were first detected at 2 (MS) or 3 (ICL) days after germination (DAG), peaked at 5 DAG, and decreased thereafter. By reverse transcriptase-polymerase chain reaction (RT-PCR), the germination-specific genes were amplified in both ABA-insensitive (vp1) and ABA-deficient (vp7 and vp10) mutant embryos at 26 and 33 days after pollination (DAP), but not in wild-type embryos. The repression of these germination-specific genes thus requires the Vp1 gene product and normal levels of ABA to induce embryo quiescence during kernel development. This suggests that a genetic regulatory system exists to prevent vivipary in developing maize embryos. The involvement of the Vp1 gene product and ABA in repressing germination-specific genes complements their previously defined roles in the induction of seed-specific genes such as C1.
Topics: Abscisic Acid; Base Sequence; Cloning, Molecular; DNA, Complementary; DNA-Binding Proteins; Gene Expression; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Germination; Isocitrate Lyase; Molecular Sequence Data; Plant Proteins; Repressor Proteins; Seeds; Sequence Homology, Amino Acid; Trans-Activators; Transcription Factors; Transcription, Genetic; Zea mays
PubMed: 9666472
DOI: No ID Found -
Plant Physiology Nov 1987Glyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720...
Glyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contamination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate of 340 nanomole electrons per minute per milligram glyoxysomal protein. Palmitoyl-CoA oxidation could be coupled to ferricyanide or cytochrome c reduction. Complete oxidation of palmitoyl-CoA, yielding 14 nanomole electrons/per nanomole palmitoyl-CoA, was demonstrated with the acceptors, NAL, cytochrome c, and ferricyanide. Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by beta-oxidation and the glyoxylate cycle.
PubMed: 16665778
DOI: 10.1104/pp.85.3.792 -
Plant Physiology Feb 1996The glyoxysomes of growing oilseed seedlings produce H2O2, a reactive oxygen species, during the beta-oxidation of lipids stored in the cotyledons. An expression library... (Comparative Study)
Comparative Study
The glyoxysomes of growing oilseed seedlings produce H2O2, a reactive oxygen species, during the beta-oxidation of lipids stored in the cotyledons. An expression library of dark-grown cotton (Gossypium hirsutm L.) cotyledons was screened with antibodies that recognized a 31-kD glyoxysomal membrane polypeptide. A full-length cDNA clone (1258 bp) was isolated that encodes a 32-kD subunit of ascorbate peroxidase (APX) with a single, putative membrane-spanning region near the C-terminal end of the polypeptide. Internal amino acid sequence analysis of the cotton 31-kD polypeptide verified that this clone encoded this protein. This enzyme, designated gmAPX, was immunocytochemically and enzymatically localized to the glyoxysomal membrane in cotton cotyledons. The activity of monodehydroascorbate reductase, a protein that reduces monodehydroascorbate to ascorbate with NADH, also was detected in these membranes. The co-localization of gmAPX and monodehydroascorbate reductase within the glyoxysomal membrane likely reflects an essential pathway for scavenging reactive oxygen species and also provides a mechanism to regenerate NAD+ for the continued operation of the glyoxylate cycle and beta-oxidation of fatty acids. Immunological cross-reactivity of 30- to 32-kD proteins in glyoxysomal membranes of cucumber, sunflower, castor bean, and cotton indicate that gmAPX is common among oilseed species.
Topics: Amino Acid Sequence; Ascorbate Peroxidases; Base Sequence; Cell Fractionation; Cyanogen Bromide; DNA Primers; DNA, Complementary; Immunoblotting; Membrane Proteins; Microscopy, Immunoelectron; Molecular Sequence Data; Organelles; Peptide Fragments; Peroxidases; Polymerase Chain Reaction; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 8742335
DOI: 10.1104/pp.110.2.589