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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed beta-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. Immunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.
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PMID:Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase. 883 11

Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypeptide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other microbody proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells.
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PMID:Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence. 955 62

Employing transgenic Arabidopsis plants, we analyzed the mechanism for the translocation of peroxisomal proteins from the cytosol into the matrix that is mediated by the N-terminal targeting signal. A hybrid Arabidopsis variety was generated which accumulates two kinds of originally bacterial proteins, beta-glucuronidase (GUS) and a GUS chimeric protein designated as CS-delta C42-GUS, that carries the N-terminal targeting signal for glyoxysomal citrate synthase. Because the CS-delta C42-GUS is targeted to peroxisomes but had never been observed to be processed to produce the mature protein, it can be distinguished from the GUS protein by its molecular size. Cell fractionation analyses showed that the native GUS protein, although lacking the targeting signal, was co-localized with the CS-delta C42-GUS protein in the peroxisomes of the hybrid plant. It is suggested that the native GUS protein forms oligomeric structures with the peroxisome-targeted chimeric proteins and can therefore be transported into peroxisomes. Sucrose density gradient centrifugation revealed that the native GUS and the chimeric GUS indeed are present both as a dimer and a tetramer in the Arabidopsis hybrid variety.
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PMID:Oligomeric proteins containing N-terminal targeting signals are imported into peroxisomes in transgenic Arabidopsis. 1048 23

The Arabidopsis thaliana gene CUT1 encodes a very-long-chain fatty acid-condensing enzyme required for the production of epicuticular wax in bolting stems. We have examined the expression pattern of CUT1 in Arabidopsis at different developmental stages and under different environmental conditions. RNA blot analysis showed that CUT1 was highly expressed in shoots, but not in roots. CUT1 expression was detectable throughout development. Light was required for CUT1 expression, and expression was increased by salt and drought treatments. The promoter region of the CUT1 gene was cloned, and 1.2 kb of the sequence 5' to the translation start codon was used to direct beta-glucuronidase (GUS) expression in transgenic plants. Histochemical and fluorometric (quantitative) GUS assays confirmed that the CUT1 promoter directed epidermal-specific expression and was highly active in Arabidopsis and in tobacco. A construct using the CUT1 promoter to drive CUT1 expression (CUT1p-CUT1) was used to transform Arabidopsis. Transgenic plants which had somewhat increased (overexpression) or greatly reduced (co-suppression) wax loads were recovered. Thus, the CUT1 promoter should be useful for genetic engineering applications that require epidermis-specific expression of genes.
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PMID:Expression of the wax-specific condensing enzyme CUT1 in Arabidopsis. 1117 Nov 58

Plant peroxisomes contain at least four proteins, namely, citrate synthase, malate dehydrogenase, long-chain acyl-CoA oxidase, and 3-ketoacyl-CoA thiolase, which are synthesized as large precursors with an N-terminal cleavable presequence. Each presequence has a conserved domain (R[I/L/Q]-X5-HL) that is homologous to peroxisomal targeting signal 2 from mammals and yeasts. In addition, a cysteine residue is found at the C-terminal ends of the presequences, whose function has not yet been described. The authors analyzed the function of the presequences and the conserved amino acids using transgenic Arabidopsis plants, which accumulate beta-glucuronidase carrying the presequence of the peroxisomal proteins from plants. Immunological and immunocytochemical studies on the transgenic plants showed that a conserved sequence in the extrapeptides is essential for targeting to peroxisomes, and a cysteine residue at the cleavage site is involved in the processing of the presequence. These results suggest that the presequences of the peroxisomal proteins function as targeting signals, and are necessary for the recognition of the processing.
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PMID:Transport of peroxisomal proteins synthesized as large precursors in plants. 1133 56

Lesquerella fendleri seed oil contains up to 60% hydroxy fatty acids, nearly all of which is the 20-carbon hydroxy fatty acid lesquerolic acid (D-14-hydroxyeicos-cis-11-enoic acid). Previous work suggested that lesquerolic acid in L. fendleri was formed by the elongation of the 18-carbon hydroxy fatty acid, ricinoleic acid. To identify a gene encoding the enzyme involved in hydroxy fatty acid elongation, an L. fendleri genomic DNA library was screened using the coding region of the Arabidopsis Fatty Acid Elongation1 gene as a probe. A gene, LfKCS3, with a high sequence similarity to known very long-chain fatty acid condensing enzymes, was isolated. LfKCS3 has a 2,062-bp open reading frame interrupted by two introns, which encodes a polypeptide of 496 amino acids. LfKCS3 transcripts accumulated only in the embryos of L. fendleri and first appeared in the early stages of development. Fusion of the LfKCS3 promoter to the uidA reporter gene and expression in transgenic Arabidopsis resulted in a high level of beta-glucuronidase activity exclusively in developing embryos. Seeds of Arabidopsis plants transformed with LfKCS3 showed no change in their very long-chain fatty acid content. However, when these Arabidopsis plants were crossed with the transgenic plants expressing the castor oleate 12-hydroxylase, significant amounts of 20-carbon hydroxy fatty acids accumulated in the seed, indicating that the LfKCS3 condensing enzyme specifically catalyzes elongation of 18-carbon hydroxy fatty acids.
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PMID:A condensing enzyme from the seeds of Lesquerella fendleri that specifically elongates hydroxy fatty acids. 1174 8

To learn more about the role of the CER6 condensing enzyme in Arabidopsis surface wax production, we determined CER6 transcription domains and the timing of CER6 transcription in vegetative and reproductive structures from juvenile, mature, and senescing tissues. We found that CER6 is highly transcribed throughout development, exclusively in the epidermal cells in all tissues examined. The only exception to the epidermal expression was observed in anthers nearing maturity, in which CER6 mRNA was localized in the tapetum. To determine if environmental factors such as light and water deficit, which are known to stimulate wax accumulation, induce CER6 transcription, we examined the effects of these factors on CER6 transcript abundance. Our results demonstrate that light is essential for CER6 transcription, and that osmotic stress and the presence of abscisic acid enhance CER6 transcript accumulation. CER6 promoter-directed expression of the beta-glucuronidase reporter gene in transgenic plants demonstrated that the CER6 promoter was highly effective in directing epidermis-specific expression in Arabidopsis and tobacco (Nicotiana tabacum). Furthermore, CER6 promoter-driven CER6 overexpression resulted in increased wax deposition in Arabidopsis stems. These experiments indicate that the expression level of CER6 in the epidermis is one of the factors controlling wax accumulation on Arabidopsis stems.
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PMID:Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. 1217 69


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