A novel tetratricopeptide repeat-containing J-protein localized in a plasma membrane-bound protein complex of the phytopathogenic oomycete Phytophthora megasperma.

Phytoalexins originating from plant tissues may cause within cells of fungi or oomycetes a change in the localization of actin, tubulin and chaperones. To test the hypothesis in a filamentously growing oomycete, we compared the distribution of cellular markers in the presence and absence of hydroxystilbene phytoalexins. Using cDNA from the phytopathogenic organism Phytophthora megasperma, the causal agent of root rot on soybean and many other plants, and including probes for Hsp70 and Hsp40, we cloned a DnaJ-protein (Jcp) with the capacity of interacting with both a particular Hsp70 isoform via its J-domain and with other proteins via its tetratricopeptide repeat (TPR) domain. Antisera raised against the bacterially expressed protein Jcp allowed the analysis of its intracellular localization during hyphal growth. Following the subfractionation of cell homogenates, we detected virtually all immunoreactive Jcp in the plasma membrane-enriched fraction and as constituent of a membrane-associated protein complex. In agreement with the biochemical findings, immunocytochemical stains of hyphae showed Jcp as part of cortical patches positioned along the plasma membrane similar to the distribution of actin patches. Confocal microscopy, however, revealed that the Jcp-containing patches did not generally co-localize with the patches visualized by the actin stain. The 59-kDa Jcp, characterized by a large 8-fold TPR domain at the N-terminal region and a J-domain close to the C-terminus, is a good candidate for bridging the gap between Hsp70 and Hsp90 by protein-protein interactions. By administration of plant-derived phytoalexins it was shown that the presence of resveratrol or piceatannol significantly reduces the amount of the Jcp-containing patches, but does not lead to a relocalization of intracellular Jcp.

Phospholipid monolayer of plant lipid bodies attacked by phospholipase A2 shows 80 nm holes analyzed by atomic force microscopy.

In plant storage tissue, lipid bodies are composed of triacylglycerides and surrounded by a phospholipid monolayer which is stabilized by oleosins. At the onset of lipid body mobilization, cells express phospholipase A2, which partially degrades the monolayer and thus provides access for the subsequently acting triacylglyceride degrading enzymes. Analyzing the lipid body surface by atomic force microscopy we show that, at the stage of maximal phospholipase A2 expression, the monolayer contains holes of approximately 80 nm in width and 2.45 +/- 0.46 nm in depth. Non-contact mode imaging was performed with a lateral resolution of approximately 10 nm and a vertical resolution of less than 0.1 nm. The depth of the holes corresponds to the width of the monolayer, while the width of the channels is sufficiently large to provide access to 100 kDa enzymes, such as lipoxygenase and lipases.

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies.

A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid.

The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.

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.

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.

Grapevine protoplasts as a transient expression system for comparison of stilbene synthase genes containing cGMP-responsive promoter elements.

A method for preparing elicitor-responsive protoplasts from grapevine cells kept in suspension culture was established. The protoplasts were employed in order to perform transient gene expression experiments produced by externally added plasmids. Using the gene coding for bacterial beta-glucuronidase as the reporter gene, the transient expression under the control of various promoters of stilbene synthase genes were analyzed. The elicitor-responsiveness of promoters from grapevine genes and heterologous promoters were assayed: the grapevine stilbene synthase gene VST-1 and pine stilbene synthase genes PST-1, PST-2 and PST-3. Compared to the expression effected by the cauliflower mosaic virus 35S RNA-promoter, the stilbene synthase promoters caused a 2-5-fold increase in GUS-activity. Incubation of transformed protoplasts with fungal cell wall further stimulated the stilbene synthase promoters but not the 35S RNA-promoter. An even more pronounced differentiation between the promoters was observed when cGMP was included in the transient expression assays. Instead of treating transformed protoplasts with fungal cell wall we administered simultaneously cGMP and the plasmid to be tested. The cGMP-responsive increase was (a) specific concerning the nucleotide applied, (b) characteristic of grapevine protoplasts, and (c) not seen with shortened promoter-GUS constructs or GUS under the control of the 35S RNA-promoter. The highest cGMP-dependent response to stress was shown by the promoter of the grapevine stilbene synthase gene VST-1.

Characterization of a pine multigene family containing elicitor-responsive stilbene synthase genes.

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial beta-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.

A phospholipase A2 is transiently synthesized during seed germination and localized to lipid bodies.

A patatin-like protein is present in the storage tissue of cucumber seedlings during the stage of fat mobilization. The cucumber protein is a homologue of a glycoprotein which in potatoes accounts for most of the total protein content of tubers. Following preparation of a cucumber cDNA library representing the developmental stage of cotyledons of 1 day old germinating seeds we isolated and characterized a clone encoding a patatin-like protein. Antibodies raised against the protein expressed in bacteria were used for immunodetection in subcellular fractions of cucumber seedlings. It was shown that the patatin-like protein was virtually exclusively confined to lipid bodies. The protein expressed in bacteria was characterized in vitro by its esterase activity acting on monoacylglycerols and phospholipids. Detailed analysis using various forms of phosphatidyl choline as substrates demonstrated that the patatin-like protein is a phospholipase A2 acting on palmitoyl, linoleoyl and hydroperoxidized linoleoyl groups equally well. Studying the temporal and tissue-specific expression of patatin-like protein mRNA we showed its appearance exclusively during fat catabolism. As maximal amounts of the protein were found at an early stage of mobilization and confined to lipid bodies, we propose that the patatin-like hydrolase is involved in lipid body mobilization.

All three acyl moieties of trilinolein are efficiently oxygenated by recombinant His-tagged lipid body lipoxygenase in vitro.

Recently, we found a 13-lipoxygenase in germinating cucumber cotyledons, which was located at the lipid body membrane. Based on its products formed mobilization of storage lipids seems to be initiated by this 13-lipoxygenase. For biochemical characterization its cDNA was expressed as His-tagged protein. Active recombinant enzyme was obtained from low temperature cultivation of E. coli after affinity purification. It (i) exhibited an unchanged region specificity, and (ii) showed a pH optimum of 7.2 against trilinolein as substrate. We compared its ability to oxygenate trilinolein with the one of another 13-lipoxygenase, soybean lipoxygenase-1. At the pH optimum of soybean lipoxygenase-1 (9.0), trilinolein was oxygenated only to 28% of the amount converted by the lipid body lipoxygenase. Moreover, trilinolein oxygenation by soybean lipoxygenase-1 leads mainly to monohydroperoxy derivatives, whereas oxygenation by lipid body LOX leads to a trihydroperoxy derivative.

Heat shock transiently enhances the synthesis rate of Sis1p, a ribosome-associated DnaJ protein in the oleagenous yeast Apiotrichum curvatum.

DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)(+)-RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39.5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p.

Plant polyketide synthases leading to stilbenoids have a domain catalyzing malonyl-CoA:CO2 exchange, malonyl-CoA decarboxylation, and covalent enzyme modification and a site for chain lengthening.

Stilbene synthases and the related bibenzyl synthases are plant polyketide synthases whose biological functions lie in the formation of antimicrobial phytoalexins. The formation of hydroxystilbenes from one molecule of acyl-CoA and three molecules of malonyl-CoA is catalyzed by a homodimeric 90 kDa protein and includes Claisen condensations and cleavage of a thioester followed by decarboxylation. Combining inhibitor studies, protein modifications, and site-directed mutagenesis, we were able to differentiate between the binding sites for malonyl-CoA and the regions responsible for the selection of the primer, p-coumaroyl-CoA or m-hydroxyphenylpropionyl-CoA, respectively. Mutations in the C-terminal part of the molecule or modification by photolabeling with p-azidocinnamoyl-CoA influence the overall reaction, the formation of hydroxystilbenes, but leave partial reactions, such as the malonyl-CoA:CO2 exchange and the malonyl-CoA-dependent modification of the enzyme, unaffected. Data obtained with several kinds of stilbene synthase and mutant forms suggest that the malonyl-CoA-dependent covalent modification takes place at a cysteine residue in the N-terminal part of the enzyme. Mutations in the C-terminal half of the enzyme molecule do not interfere with the malonyl-CoA-dependent reactions.

Lipid body lipoxygenase characterized by protein fragmentation, cDNA sequence and very early expression of the enzyme during germination of cucumber seeds.

Lipid bodies are cellular compartments containing triacylglycerols. They are encompassed by a phospholipid monolayer and decorated with characteristic proteins. In plants, lipid bodies are synthesized during seed formation but acquire new proteins during seed germination. In germinating cucumber (Cucumis sativus) seeds, the set of newly synthesized proteins appearing in the lipid bodies at the early stage of triacylglycerol mobilization comprises a special form of lipoxygenase. We isolated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogen bromide. A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lipoxygenase forms during germination. This allowed a clear distinction of this enzyme from other lipoxygenase isoforms. Hence, for determining the molecular structure of lipid body lipoxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day 1 of germination. From the cDNA sequence, oligonucleotides were derived that specifically detected lipid body lipoxygenase mRNA on northern blots. The very early expression of lipid body lipoxygenase was corroborated by this approach. Good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically. In particular, the cleavage products of cyanogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA. The sequence data show a lipoxygenase form characterized by a molecular mass of 99655 Da, which is significantly higher than the molecular masses of the cytosolic forms. Compared to the cytosolic forms that exhibit a molecular mass of 95 kDa, the lipid body form has an N-terminal extension of 34 amino acid residues. No evidence for a cotranslational or post-translational proteolytic processing was obtained by the size comparison of the in vitro-translated lipoxygenase and the lipid body form.

Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls.

T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP-1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species.

Lipoxygenase-catalyzed oxygenation of storage lipids is implicated in lipid mobilization during germination.

The etiolated germination process of oilseed plants is characterized by the mobilization of storage lipids, which serve as a major carbon source for the seedling. We found that during early stages of germination in cucumber, a lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.11.12) form is induced that is capable of oxygenating the esterified fatty acids located in the lipid-storage organelles, the so-called lipid bodies. Large amounts of esterified (13S)-hydroxy-(9Z,11E)-octadecadienoic acid were detected in the lipid bodies, whereas only traces of other oxygenated fatty acid isomers were found. This specific product pattern confirms the in vivo action of this lipoxygenase form during germination. Lipid fractionation studies of lipid bodies indicated the presence of lipoxygenase products both in the storage triacylglycerols and, to a higher extent, in the phospholipids surrounding the lipid stores as a monolayer. The degree of oxygenation of the storage lipids increased drastically during the time course of germination. We show that oxygenated fatty acids are preferentially cleaved from the lipid bodies and are subsequently released into the cytoplasm. We suggest that they may serve as substrate for beta-oxidation. These data suggest that during the etiolated germination, a lipoxygenase initiates the mobilization of storage lipids. The possible mechanisms of this implication are discussed.

Fatty acid beta-oxidation in glyoxysomes. Characterization of a new tetrafunctional protein (MFP III).

We describe a new form of a multifunctional protein possessing the enzyme activities of delta 3, delta 2-enoyl-CoA isomerase, 3-hydroxyacyl-CoA epimerase, L-3-hydroxyacyl-CoA dehydrogenase and L-3-hydroxyacyl-CoA forming 2-trans-enoyl-CoA hydratase. This isoform, characterized by a molecular mass of 81 kDa and an isoelectric point above pH 9, was designated MFP III. Along with the tetrafunctional 76.5 kDa MFP II and the trifunctional 74 kDa MFP I, MFP III participates in degradation of fatty acid in glyoxysomes during mobilization of fat reserves. In combination with thiolase, MFP III encompasses all activities to degrade 3-cis-enoyl-CoAs to acetyl-CoA.

The inducible 9, 10-dihydrophenanthrene pathway: characterization and expression of bibenzyl synthase and S-adenosylhomocysteine hydrolase.

Tricyclic 9,10-dihydrophenanthrenes originate from phenylpropane derivatives by chain elongation and cyclization according to the polyacetate rule. Bibenzyls are bicyclic intermediates, and O-methylation is a prerequisite for their conversion into dihydrophenanthrenes. cDNA clones encoding bibenzyl synthases and S-adenosylhomocysteine hydrolase of the orchid Phalaenopsis sp. were isolated from a cDNA library representing the stage of elicitor-induced plants. The deduced amino acid sequences of two clones, pBibSy811 and pBibSy212, indicated that we obtained two full-length sequences of bibenzyl synthases characterized by their homology to stilbene synthases previously investigated. That indeed bibenzyl synthase cDNAs rather than a homologous stilbene synthase cDNA or chalcone synthase cDNA have been isolated was demonstrated by expression of two enzymatically active bibenzyl synthase proteins in Escherichia coli. These proteins showed virtually the same selectivity towards m-hydroxyphenylpropionyl-CoA as substrate as the enzyme isolated from orchid plants. In young sterile Phalaenopsis plants, the formation of both bibenzyl synthase mRNAs and S-adenosylhomocysteine hydrolase mRNAs was increased upon elicitation more than 100-fold. The time courses of gene expression exhibited transient profiles, reaching maximum mRNA levels 20 h after onset of fungal infection followed by a rapid decline to 40 h.

Evidence for domain structures of the trifunctional protein and the tetrafunctional protein acting in glyoxysomal fatty acid beta-oxidation.

In plant glyoxysomes, an enzyme activity responsible for a particular step in the fatty acid beta-oxidation is located on more than one protein species. Various monofunctional enzymes and two forms of a multifunctional protein are involved in the degradation of cis-unsaturated fatty acids. delta 3, delta 2-Enoyl-CoA isomerase activity, previously found to be located on a monofunctional dimeric protein, is attributable to one form of the monomeric multifunctional protein (MFP). The presence or absence of isomerase activity allows us to differentiate between the tetrafunctional 76.5-kDa isoform (MFP II) and the trifunctional 74-kDa isoform (MFP I) in cucumber (Cucumis sativus) cotyledons. Both MFP I and MFP II exhibited blocked N-terminal structures. MFP I and MFP II are distinguishable from each other by their susceptibility to limited proteolysis. A series of examples is presented describing the preparation of enzymically active proteolytic fragments. We demonstrate that both forms of the monomeric MFP are composed of domains separable from each other without loss of activity. By fragmentation of MFP I and subsequent chromatography, a 60-kDa peptide was purified retaining hydratase and epimerase activity but lacking dehydrogenase activity. In addition, a highly positively charged fragment was observed carrying solely dehydrogenase activity. From MFP II, a 36-kDa fragment with hydratase activity was characterized. An enzymically inactive 46-kDa fragment was prepared from MFP II and sequenced at its unblocked N-terminus.

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment.

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3' end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5' flanking region of gene Vst1 contains a TATAA box at nucleotide -48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.

Domains of the tetrafunctional protein acting in glyoxysomal fatty acid beta-oxidation. Demonstration of epimerase and isomerase activities on a peptide lacking hydratase activity.

Peroxisomes from different eukaryotic organisms house a multifunctional protein acting in fatty acid beta-oxidation. In plant glyoxysomes, one of the isoforms of this protein contains the activities of L-3-hydroxyacyl-CoA hydrolyase (EC 4.2.1.17), L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.211), D-3-hydroxyacyl-CoA epimerase, and delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8). This was demonstrated after molecular cloning of a cDNA coding for a protein of 79047 Da and its bacterial expression. Chromatographic purification yielded a monomeric protein exhibiting all four activities. In addition, mutant forms were prepared, and peptides representing single domains were purified. Peptides containing the N-terminal region showed D-3-hydroxyacyl-CoA epimerase and delta 3,delta 2-enoyl-CoA isomerase activities but lacked 2-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities. Using the N-terminal fragment, we demonstrated that the D-3-hydroxyacyl-CoA converting activity is actually an epimerase rather than part of a combined water eliminating and water attaching system. The C-terminal half of the multifunctional protein represents the dehydrogenase domain.

Heat shock enhances the amount of prenylated Dnaj protein at membranes of glyoxysomes.

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.