Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function.
Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function.
复制标题
DOI：
10.1016/s0021-9258(18)53392-5
复制
发表时间：
1993-03
期刊：
影响因子：
--
通讯作者：
V. Esser;C. Britton;B. Weis;D. W. Foster;J. McGarry
中科院分区：
文献类型：
--
作者：
V. Esser;C. Britton;B. Weis;D. W. Foster;J. McGarry
我们报告了编码大鼠肝脏肉毒碱棕榈酰转移酶 I (CPT I) 的全长 cDNA 的分离和表征。对应于源自大鼠肝线粒体的丙二酰辅酶A/依托莫克西辅酶A结合蛋白的两种胰蛋白酶肽的寡核苷酸（Esser，V.，Kuwajima，M.，Britton，C.H.，Krishnan，K.，Foster，D.W.，和McGarry，J.D.(1993) J.Biol.Chem.268, 5810-5816)用于筛选在质粒克隆载体pcDV中构建的大鼠肝脏cDNA文库。获得的克隆由 102 个核苷酸的 5'-非翻译区、预测 773 个氨基酸的蛋白质 (M(r) = 88,150) 的 2,319 个碱基的单个开放阅读框、以及后面跟着 Poly(A)+ 尾的 1,957 个核苷酸的 3'-非翻译片段组成。 cDNA 的 0.9 千碱基片段识别了大鼠肝脏中的单一 mRNA 种类（大小约为 4.7 千碱基）。 cDNA 的身份通过以下发现得到证实：(i) 开放阅读框编码原始蛋白质中发现的所有四种肽； (ii) 用亚克隆到表达载体 pCMV6 中的 cDNA 转染 COS 细胞，导致丙二酰辅酶 A 和依托莫克西辅酶 A 敏感的 CPT 活性选择性诱导 10-20 倍； (iii)过表达的产物很容易通过针对起始材料产生的抗体在蛋白质印迹上检测到。从头合成的酶似乎有可能通过前导肽靶向线粒体外膜，并且成熟蛋白通过其氨基末端附近的一段 20 个氨基酸实现膜锚定。该蛋白质的预测氨基酸序列显示出与其他三种大鼠酰基转移酶（即肝 CPT II、肝肉碱辛酰基转移酶和脑胆碱乙酰基转移酶）的氨基酸序列具有很强同一性的区域。这些发现首次深入了解了 CPT I 同工型的结构。他们还明确证实 CPT I 和 CPT II 是不同的蛋白质，并且 CPT I 抑制剂在其催化结构域内相互作用，而不是与相关的调节成分相互作用。
We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5‘-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (M(r) = 88,150), and a 3‘-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (approximately 4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.