Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria.
L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
Bourguignon
J
J
Laboratoire de Physiologie Cellulaire Végétale, Unit Associé au Centre National de la Recherche Scientifique No 576, Grenoble, France.
Macherel
D
D
Neuburger
M
M
Douce
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D007887:Fabaceae / Q000201:enzymology*; D015870:Gene Expression; D005980:Glutathione Reductase / Q000235:genetics; D005998:Glycine / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008027:Light; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment
D011768:Pyruvate Dehydrogenase Complex; D012333:RNA, Messenger; D004247:DNA; D008058:Dihydrolipoamide Dehydrogenase; D005980:Glutathione Reductase; D005998:Glycine