Enzymatic methylation of in vitro synthesized apocytochrome c enhances its transport into mitochondria.
Enzymatic methylation of in vitro synthesized apocytochrome c enhances its transport into mitochondria.
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体外合成的脱辅基细胞色素 c 的酶促甲基化增强了其向线粒体的转运。
DOI：
10.1016/s0021-9258(18)47852-0
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Woon Ki Paik
中科院分区：
文献类型：
--
作者：
Kwang Sook Park;B. Frost;M. Tuck;Ling Ling;Sangduk Kim;Woon Ki Paik
将酿酒酵母的iso1 -细胞色素c基因克隆到含有活性噬菌体SP启动子的pSP65载体中。克隆到的iso1 -细胞色素c基因为856碱基对的XhoI-HindIII片段。当质粒在基因终止密码子下游279个碱基的HindIII位点被酶切后，用SP6 RNA聚合酶在体外转录，得到全长转录本。利用兔网织细胞裂解液系统翻译SP6 - iso1 -cytochrome c mRNA，并用十二烷基硫酸钠-聚丙烯酰胺凝胶分析蛋白质产物。自荧光法检测到一个分子量为12000的主要条带，与酿酒酵母细胞色素c的考马斯氏染色条带相吻合。该产品在等电聚焦凝胶上也被证明与标准酵母细胞色素c相同。在体外翻译混合物中加入部分纯化的s -腺苷基-l -蛋氨酸和s -腺苷基-l -蛋氨酸，对体外合成的异-1-apocytochrome c进行酶化甲基化。甲基化被甲基化酶抑制剂s -腺苷- l-同型半胱氨酸或蛋白质合成抑制剂嘌呤霉素抑制。蛋白质中甲基化氨基酸的主要类型为epsilon- n-三甲基赖氨酸，占总甲基化氨基酸的77%。最后，体外合成的iso1 -apocytochrome c的甲基化使其进入酿酒酵母分离的线粒体的量比未甲基化的蛋白增加2-4倍，但不进入大鼠肝脏线粒体。这表明甲基化通过一种特定的受体机制促进了细胞色素c进入线粒体。
The gene for iso-1-cytochrome c from Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP promoter. The iso-1-cytochrome c gene was cloned as an 856-base pair XhoI-HindIII fragment. When the resulting plasmid was digested at the HindIII site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system, and the protein products were analyzed on sodium dodecyl sulfate-polyacrylamide gels. One major band with a molecular weight of 12,000 was detected by autofluorography and coincided with the Coomassie staining band of apocytochrome c from S. cerevisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectrofocusing gel. The in vitro synthesized iso-1-apocytochrome c was enzymatically methylated by adding partially purified S-adenosyl-L-methionine:cytochrome c-lysine N-methyltransferase (protein methylase III, EC 2.1.1.59) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor puromycin. The principal type of methylated amino acid in the protein was found to be epsilon-N-trimethyllysine which accounted for 77% of the total. Finally, the methylation of in vitro synthesized iso-1-apocytochrome c was found to increase its import into mitochondria isolated from S. cerevisiae 2-4-fold over unmethylated protein, but not into rat liver mitochondria. This suggests that methylation facilitates the import of apocytochrome c into mitochondria by a specific receptor mechanism.