Evidence for multiple forms of biotin holocarboxylase synthetase in pea (Pisum sativum) and in Arabidopsis thaliana: subcellular fractionation studies and isolation of a cDNA clone.
The intracellular compartmentation of biotin holocarboxylase synthetase has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in cytosol (approx. 90% of total cellular activity). Significant activity was also identified in the soluble phase of both mitochondria and chloroplasts. Two enzyme forms were separated by anion-exchange chromatography. The major form was found to be specific for the cytosol compartment, whereas the minor form was present in mitochondria as well as in chloroplasts. We also report the isolation and DNA sequence of a cDNA encoding an Arabidopsis thaliana biotin holocarboxylase synthetase. This cDNA was isolated by functional complementation of a conditional lethal Escherichia coli birA (biotin ligase gene, which regulates biotin synthesis) mutant. This indicated that the recombinant plant protein was able to biotinylate specifically an essential apoprotein substrate in the bacterial host, that is a subunit of acetyl-CoA carboxylase called biotin carboxyl carrier protein. The full-length nucleotide sequence (1534 bp) encodes a protein of 367 amino acid residues with a molecular mass of 41172 Da and shows specific regions of similarity to other biotin holocarboxylase synthetase genes as isolated from bacteria and yeast, and with cDNA species from human. A sequence downstream of the first translation initiation site encodes a putative peptide structurally similar to organelle-targeting pre-sequences, suggesting a mitochondrial or chloroplastic localization for this isoform.
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Laboratoire Mixte CNRS/Rhône-Poulenc (UM41 associée au Centre National de la Recherche Scientifique), Rhône-Poulenc Agrochimie, 14-20 rue Pierre Baizet, 69263 Lyon cedex 9, France.
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D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D015139:Blotting, Southern; D019731:Carbon-Nitrogen Ligases; D002645:Chickens; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D014176:Protein Biosynthesis; D013347:Subcellular Fractions / Q000201:enzymology; D014158:Transcription, Genetic
D018076:DNA, Complementary; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases