Costantino P, Attardi G
J Biol Chem. 1977 Mar 10;252(5):1702-11.
The metabolic behavior of the mitochondrial protein synthesis products has been investigated in HeLa cells. Particular attention was given to the four major electrophoretic components (designated as Nos. 2, 3, 5, and 8) of the 10 previously identified as organelle-specific products. Inhibition of cytoplasmic protein synthesis with emetine or cycloheximide causes a rapid decline in the rate of mitochondrial protein synthesis, with an estimated half-life of 1 to 2 h, affecting in a parallel way all the discrete components. About 30% of the original synthetic activity appears to be resistant to emetine treatment for at least 24 h; however, all the polypeptides synthesized after the first 4 h of cell exposure to emetine are metabolically unstable, possibly because of lack of integration into the inner mitochondrial membrane. An analysis of the stability of newly synthesized products of mitochondrial protein synthesis pulse-labeled in the presence of cycloheximide and then chased in the absence of the drug (i.e. under conditions of resumed cytoplasmic protein synthesis) has revealed marked differences among the various discrete components. In particular, about three-fourths of the radioactivity associated with components 3 and 5 decays within 4 h of chase, the remainder being substantailly stable afterwards; by contrast, the radioactivity in components 2 and 8 shows only a slow decline during a 3-day chase. If the chase is carried out under conditions of a persistent block of cytoplasmic protein synthesis, as is the situation after a pulse labeling in the presence of emetine, all newly synthesized components appear to be destablized in various degrees, with the exception of component 5, which is to a great extent stabilized. Inhibition of mitochondrial protein synthesis with chloramphenicol has a progressive stabilizing effect on most of the discrete components newly synthesized after removal of the drug; this effect is especially striking in the case of component 5 which, in experiments of continuous labeling in the presence of emetine after prolonged chloramphenicol treatment, becomes, after 24 h of labeling or more, the only recognizable peak in the electrophoretic pattern of the sodium dodecyl sulfate-lysed mitochondrial fraction. The results of the kinetic experiments described here are interpreted in terms of two roles of cytoplasmically synthesized proteins, one required for the synthesis of polypeptides within the organelles, the other necessary for the stabilization of the mitochondrial products.
已在HeLa细胞中研究了线粒体蛋白质合成产物的代谢行为。特别关注了先前鉴定为细胞器特异性产物的10种中的4种主要电泳成分（编号为2、3、5和8）。用依米丁或环己酰亚胺抑制细胞质蛋白质合成会导致线粒体蛋白质合成速率迅速下降，估计半衰期为1至2小时，以平行方式影响所有离散成分。约30%的原始合成活性似乎对依米丁处理至少24小时具有抗性；然而，细胞暴露于依米丁后最初4小时后合成的所有多肽在代谢上不稳定，可能是因为缺乏整合到线粒体内膜中。对在环己酰亚胺存在下脉冲标记然后在无药物情况下追踪（即在恢复细胞质蛋白质合成的条件下）的线粒体蛋白质合成新合成产物的稳定性分析揭示了各种离散成分之间的显著差异。特别是，与成分3和5相关的放射性约四分之三在追踪4小时内衰减，其余部分此后基本稳定；相比之下，成分2和8中的放射性在3天追踪期间仅缓慢下降。如果在细胞质蛋白质合成持续受阻情况下进行追踪（就像在依米丁存在下脉冲标记后的情况），所有新合成成分似乎都在不同程度上不稳定，但成分5除外，它在很大程度上是稳定的。用氯霉素抑制线粒体蛋白质合成对去除药物后新合成的大多数离散成分具有逐渐稳定的作用；这种作用在成分5的情况下尤为显著，在长期氯霉素处理后在依米丁存在下进行连续标记的实验中，标记24小时或更长时间后，它成为十二烷基硫酸钠裂解的线粒体部分电泳图谱中唯一可识别的峰。此处描述的动力学实验结果根据细胞质合成蛋白质的两种作用进行解释，一种是细胞器内多肽合成所必需的，另一种是线粒体产物稳定所必需的。
