Molecular Cloning: and Characterization of the cDNA Coding for thCBiotin-Containing Subunit of the Chloroplastic Acetyl-Coenzyme A Carboxylase'

We report the molecular cloning and sequence of the cDNA coding for the biotin-containing subunit of the chloroplastic acetylcoenzyme A (COA) carboxylase (ACCase) of Arabidopsis tbaliana (CAC7). l h e 3' end of the CAC7 sequence, coding for a peptide of 94 amino acids, which includes a putative biotinylation motif, was expressed in Escbericbia coli as a glutathione-S-transferase (CST) fusion protein. The resulting CST-CAC1 fusion protein was biotinylated in vivo, indicating that CACl codes for a biotin-containing protein. Antibodies generated to the CST-CAC1 protein reacted solely with the 38-kD biotin-containing polypeptide of Arabidopsis. Furthermore, these antibodies inhibited ACCase activity in extracts from Arabidopsis leaves. The deduced amino acid sequence of CACl has an apparent N-terminal chloroplast-targeting transit peptide. l h e CACl protein is coded by a single Arabidopsis gene, and its mRNA accumulates to the highest levels in organs that are undergoing rapid growth. l h e amino acid sequence of the CACl protein is most similar to the biotin carboxyl-carrier protein component of eubacterial ACCases. lhese characterizations identify CACl as the biotin-containing subunit of the plastidic, heteromeric ACCase of Arabidopsis. l h e results support the ancient origin of the two structurally distinct ACCases of plants.