MOLECULAR-CLONING AND CHARACTERIZATION OF THE CDNA CODING FOR THE BIOTIN-CONTAINING SUBUNIT OF THE CHLOROPLASTIC ACETYL-COENZYME-A CARBOXYLASE

We report the molecular cloning and sequence of the cDNA coding for th e biotin-containing subunit of the chloroplastic acetyl-coenzyme A (Co A) carboxylase (ACCase) of Arabidopsis thaliana (CAC1). The 3' end of the CAC1 sequence, coding for a peptide of 94 amino acids, which inclu des a putative biotinylation motif, was expressed in Escherichia coli as a glutathione-S-transferase (CST) fusion protein. The resulting CST -CAC1 fusion protein was biotinylated in vivo, indicating that CAC1 co des for a biotin-containing protein. Antibodies generated to the CST-C AC1 protein reacted solely with the 38-kD biotin-containing polypeptid e of Arabidopsis. Furthermore, these antibodies inhibited ACCase activ ity in extracts from Arabidopsis leaves. The deduced amino acid sequen ce of CAC1 has an apparent N-terminal chloroplast-targeting transit pe ptide. The CAC1 protein is coded by a single Arabidopsis gene, and its mRNA accumulates to the highest levels in organs that are undergoing rapid growth. The amino acid sequence of the CAC1 protein is most simi lar to the biotin carboxyl-carrier protein component of eubacterial AC Cases. These characterizations identify CAC1 as the biotin-containing subunit of the plastidic, heteromeric ACCase of Arabidopsis. The resul ts support the ancient origin of the two structurally distinct ACCases of plants.