Antibodies against pex14p block ATP-independent binding of matrix proteins to peroxisomes in vitro. (1/15)

The membrane protein Pex14p is a key component of the protein import machinery of peroxisomes. Antibodies raised against human Pex14p recognise a 66 kDa protein in sunflower glyoxysomes (HaPex14p) and immunoprecipitate in vitro-translated Arabidopsis Pex14p (AtPex14p). These antibodies inhibit the ATP-independent binding to sunflower peroxisome membranes of peroxisome targeting signal type (PTS) 1- and PTS2-targeted matrix proteins, but not an integral membrane protein. These results suggest that Pex14p functions before the ATP-dependent step of peroxisome assembly.  (+info)

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. (2/15)

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.  (+info)

The glyoxylate cycle in an arbuscular mycorrhizal fungus. Carbon flux and gene expression. (3/15)

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.  (+info)

Molecular characterization of an Arabidopsis acyl-coenzyme a synthetase localized on glyoxysomal membranes. (4/15)

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.  (+info)

Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes. (5/15)

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.  (+info)

Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis. (6/15)

Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.  (+info)

Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence. (7/15)

Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D(t)) at temperatures between 4 and 32 degrees C resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K(m) for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 A. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 A. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.  (+info)

A eukaryote without catalase-containing microbodies: Neurospora crassa exhibits a unique cellular distribution of its four catalases. (8/15)

Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3'-diaminobenzidine and H(2)O(2) did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within microbodies.  (+info)