Lipoic acid metabolism in Arabidopsis thaliana: cloning and characterization of a cDNA encoding lipoyltransferase.

Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism. In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity. We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes. This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity. The expressed enzyme in the E. coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex. Comparison of the deduced amino acid sequence of LIP2 with those of E. coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria. Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves. Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A. thaliana.     

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.     

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs. A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana. At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs. Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14[C]oleoyl-CoA. A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1. Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa. The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique. These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved. Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower. Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis.     

Isolation of a cDNA encoding an Arabidopsis galactokinase by functional expression in yeast

A cDNA clone encoding Arabidopsis thaliana galactokinase was fortuitously isolated during the course of a screen for plant homologues of a Saccharomyces cerevisiae peroxisome assembly gene, PAS9. Clones were sought which restored the ability of pas9 cells to grow on oleate as a sole carbon source, as oleate metabolism requires peroxisomal -oxidation and therefore functional peroxisomes. Subsequent experiments showed that high level expression of the galactokinase cDNA did not complement the peroxisomal assembly defect, but instead permitted the cells to grow on agar plates in the absence of an external carbon source. Agar plates were shown to contain a small amount of galactose released from the agar as a result of autoclaving. The galactokinase clone was shown to be functional, as it could complement a S. cerevisiae galactokinase mutant. Galactokinase is a single copy gene in Arabidopsis, which has been designated AGK1, and is expressed in all the major organs of the plant.     

Functional cloning of an Arabidopsis thaliana cDNA encoding cycloeucalenol cycloisomerase

Plants and certain protists use cycloeucalenol cycloisomerase (EC ) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein. We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.     

Identification of a cDNA encoding an isoform of human CTP synthetase

A full-length cDNA clone encoding an isoform of human CTP synthetase (type II) was isolated. A 1761-nucleotide open reading frame which corresponds to a protein of 586 amino acids with a predicted molecular mass of 65678 Da was identified. The predicted protein sequence showed 74% identity with the translation product of a previously identified human CTP synthetase cDNA clone (type I). The function of the human cDNA encoding type II CTP synthetase was verified by successful complementation of the cytidine-requiring CTP synthetase deficient mutant JF618 of Escherichia coli. The gene encoding type II CTP synthetase has been localized on chromosome Xp22.     

Identification and characterization of a cDNA encoding a dolichyl pyrophosphate phosphatase located in the endoplasmic reticulum of mammalian cells

The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J. S., Toke, D. A., Han, G., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphatase associated with crude microsomal fractions. Mutations in CWH8 result in the accumulation of Dol-P-P, deficiency in lipid intermediate synthesis, defective protein N-glycosylation, and a reduced growth rate. A cDNA (DOLPP1, GenBank accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement the defects in growth and protein N-glycosylation, and to correct the accumulation of Dol-P-P in the cwh8Delta yeast mutant. Northern blot analyses demonstrate a wide distribution of the DOLPP1 mRNA in mouse tissues. Overexpression of Dolpp1p in yeast, COS, and Sf9 cells produces substantial increases in Dol-P-P phosphatase activity but not in dolichyl monophosphate or phosphatidic acid phosphatase activities in microsomal fractions. Subcellular fractionation and immunofluorescence studies localize the enzyme encoded by DOLPP1 to the endoplasmic reticulum of COS cells. The results of protease sensitivity studies with microsomal vesicles from the lpp1Delta/dpp1Delta yeast mutant expressing DOLPP1 are consistent with Dolpp1p having a luminally oriented active site. The sequence of the DOLPP1 cDNA predicts a polypeptide with 238 amino acids, and a new polypeptide corresponding to 27 kDa is observed when DOLPP1 is expressed in yeast, COS, and Sf9 cells. This study is the first identification and characterization of a cDNA clone encoding an essential component of a mammalian lipid pyrophosphate phosphatase that is highly specific for Dol-P-P. The specificity, subcellular location, and topological orientation of the active site described in the current study strongly support a role for Dolpp1p in the recycling of Dol-P-P discharged during protein N-glycosylation reactions on the luminal leaflet of the endoplasmic reticulum in mammalian cells.     

Identification of an Arabidopsis thaliana gene for cardiolipin synthase located in mitochondria

Cardiolipin (CL) is an anionic phospholipid with a dimeric structure. In eukaryotes, it is primarily localized in the inner membranes of mitochondria. Although the biosynthetic pathway of CL is well known, the gene for CL synthase has not been identified in any higher organisms. In this study, the CLS gene for a CL synthase has been identified in a higher plant, Arabidopsis thaliana. We have shown that the CLS gene encodes a CL synthase by demonstrating its ability to catalyze the reaction of CL synthesis from CDP-diacylglycerol and phosphatidylglycerol, and that CLS is targeted into mitochondria. These findings demonstrate that CLS is a CL synthase located in mitochondria.     

Identification of an Arabidopsis thaliana cDNA encoding a HSP70-related protein belonging to the HSP110/SSE1 subfamily

Heat-shock protein 70 (HSP70)-related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily. We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91. At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome. HSP91 is constitutively expressed in non-stressed Arabidopsis plants and is transiently induced by heat shock.     

Characterization and functional investigation of an Arabidopsis cDNA encoding a homologue to the d-PGMase superfamily

An Arabidopsis thaliana cDNA (At-74) has been isolated that encoded an uncharacterized protein showing homology with members of the d-PGMase superfamily: cofactor-dependent phosphoglycerate mutases (d-PGM-ases) and the phosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (6PF2Kase/F2, 6Pase). Preliminary phylogenetic studies indicated that At-74 cDNA and its close homologue in Arabidopsis, At-74H, belong, however, to an equally distinct group. At-74 was ubiquitously expressed in vegetative organs and induced by glucose. The At-74 cDNA was overexpressed in A. thaliana to investigate its function, but this overexpression did not result in a clear phenotype. Enzymatic assays performed on At-74-overproducing transgenic plants or E. coli cells showed no increase in either the activities of cofactor-dependent and -independent phosphoglycerate mutases (i-PGMases) and F2,6Pase or that of acid phosphatases. The possible role of At-74 in plant metabolism was further investigated by carbon partitioning experiments with [U-(14)C] glucose and measurements of soluble sugars in both young leaves and roots. Two overexpressing At-74 lines showed a clear increase in glucose uptake. This paper introduces the At-74 homologue of the d-PGMase superfamily members and supports a possible role of At-74 in carbohydrate metabolism.     

Gene note. Cloning of an Arabidopsis cDNA encoding a chloroplast localizing sulphate transporter isoform

A new member of the sulphate transporter gene in Arabidopsis encoding a chloroplast-localizing isoform, AST82 (accession no. AB008782) has been identified. AST82 had a sequence similarity with a cyanobacterial genome sequence, slr1776, which encodes a putative eukaryotic proton/sulphate co-transporter. Fusion proteins of AST82 and green fluorescent protein (GFP) transiently expressed in Arabidopsis leaves localized in chloroplasts, indicating that AST82 encodes a chloroplastic isoform.     

Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.     

Cloning and characterization of a cDNA encoding serine palmitoyltransferase in Arabidopsis thaliana

The first and committed step in de novo sphingolipid synthesis is catalysed by serine palmitoyltransferase (EC 2.3.1.50), which condenses serine and palmitoyl-CoA to form 3-ketosphinganine in a pyridoxal-5'-phosphate-dependent reaction. We have isolated and characterized a cDNA clone from Arabidopsis thaliana that is homologous to yeast and mammalian LCB2. For a functional identification, the A. thaliana homologous cDNA was expressed in Escherichia coli, which resulted in significant production of new sphinganine in E. coli cells.     

Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.     

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana

We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.