Transfer of overexpressed protein into organelle and its regulation mechanism

克隆了线粒体超长链酰基辅酶a脱氢酶cDNA。与其他酰基辅酶a脱氢酶相比，氨基酸序列表明该酶在羧基端多了约180个氨基酸多肽。在牛痘病毒中插入一系列从羧基末端缺失约60个氨基酸的多肽突变体，构建重组病毒。采用免疫印迹法检测大鼠肝癌H4的表达蛋白。在表达后的一段时间内，突变蛋白在线粒体中的稳定性不如成熟蛋白。突变蛋白也失去了膜结合和结合形成二聚体的特征。这些结果表明，羧基侧的额外多肽在线粒体中具有稳定蛋白质的独特功能。关于过氧化物酶体酰基辅酶a氧化酶羧基侧的额外多肽也得到了类似的结果。

cDNA encoding mitochondrial very-long-chain acyl-CoA dehydrogenase was cloned. Amino acid sequence indicated that the enzyme had about 180 amino acids of extra polypeptide at the carboxyl terminal side, when it was compared with the other acyl-CoA dehydrogenases. A series of mutants, deleting about 60 amino acids of polypeptide from carboxyl terminus, were inserted into vaccinia virus to construct recombinant viruses. Expressed protein in rat hepatoma H4 was examined by means of immunoblot analysis. Mutant protein was less stable in mitochondria than mature protein concerning period after the expression. Mutant protein also lost the features of membrane binding and association to form dimer. These results indicate that the extra polypeptide at carboxyl side has unique function to stabilize the protein in mitochondria. Similar results are obtained concerning the extra polypeptide at carboxyl side of peroxisomal acyl-CoA oxidase.