Amaya Y, Arakawa H, Takiguchi M, Ebina Y, Yokota S, Mori M
Institute for Medical Genetics, Kumamoto University Medical School, Japan.
J Biol Chem. 1988 Oct 5;263(28):14463-70.
Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.
大鼠3-氧代酰基辅酶A硫解酶是脂肪酸β-氧化循环中的一种酶，位于线粒体基质中。与大多数线粒体基质蛋白不同，硫解酶在合成时没有短暂的前导序列，并且在397个氨基酸残基的成熟蛋白中包含线粒体靶向和导入信息。编码硫解酶不同部分的cDNA序列与编码大鼠鸟氨酸转氨甲酰酶成熟部分（缺少其自身前导序列）的cDNA框内融合。将融合基因转染到COS细胞中，并用洋地黄皂苷进行细胞分级分离，分析融合蛋白的亚细胞定位。当表达鸟氨酸转氨甲酰酶的成熟部分时，它存在于可溶性部分中。另一方面，含有硫解酶NH2末端392、161或61个氨基酸残基的融合蛋白存在于颗粒部分中，而含有COOH末端331个残基（第62-392位残基）的融合蛋白存在于可溶性部分中。使用抗鸟氨酸转氨甲酰酶抗体进行的酶免疫细胞化学和免疫电子显微镜分析显示，含有硫解酶NH2末端部分的融合蛋白定位于线粒体。这些结果表明，大鼠3-氧代酰基辅酶A硫解酶的NH2末端61个氨基酸作为该酶蛋白线粒体靶向和导入的不可切割信号发挥作用。脉冲追踪实验表明，鸟氨酸转氨甲酰酶前体和硫解酶从细胞质进入线粒体的半衰期小于5分钟，而三种融合蛋白的半衰期为10-15分钟。有趣的是，在表达融合蛋白的细胞中，线粒体呈现异常形状，并充满免疫金阳性的晶体结构。
