Choi J K, Yu F, Wurtele E S, Nikolau B J
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Plant Physiol. 1995 Oct;109(2):619-25. doi: 10.1104/pp.109.2.619.
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 thaliana (CAC1). The 3' end of the CAC1 sequence, coding for a peptide of 94 amino acids, which includes a putative biotinylation motif, was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The resulting GST-CAC1 fusion protein was biotinylated in vivo, indicating that CAC1 codes for a biotin-containing protein. Antibodies generated to the GST-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 CAC1 has an apparent N-terminal chloroplast-targeting transit peptide. 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 similar to the biotin carboxyl-carrier protein component of eubacterial ACCases. These characterizations identify CAC1 as the biotin-containing subunit of the plastidic, heteromeric ACCase of Arabidopsis. The results support the ancient origin of the two structurally distinct ACCases of plants.
我们报道了拟南芥叶绿体乙酰辅酶A（CoA）羧化酶（ACCase）含生物素亚基（CAC1）的cDNA的分子克隆及序列。编码94个氨基酸肽段的CAC1序列的3'端，其中包括一个假定的生物素化基序，在大肠杆菌中作为谷胱甘肽-S-转移酶（GST）融合蛋白表达。所得的GST-CAC1融合蛋白在体内被生物素化，表明CAC1编码一种含生物素的蛋白。针对GST-CAC1蛋白产生的抗体仅与拟南芥中38-kD含生物素的多肽发生反应。此外，这些抗体抑制了拟南芥叶片提取物中的ACCase活性。CAC1推导的氨基酸序列具有明显的N端叶绿体靶向转运肽。CAC1蛋白由单个拟南芥基因编码，其mRNA在快速生长的器官中积累到最高水平。CAC1蛋白的氨基酸序列与真细菌ACCase的生物素羧基载体蛋白成分最为相似。这些特征确定CAC1为拟南芥质体异源ACCase含生物素的亚基。结果支持了植物中两种结构不同的ACCase的古老起源。
