TARGETING OF ORGANELLAR PROTEIN SYNTHESIS FACTORS

真核细胞中的细胞器靶向问题是 通过研究密切相关的分类来解决， 细胞质合成的多肽到线粒体和 豌豆的叶绿体。假设是 测试结果是存在某种歧视 阻止线粒体蛋白进口的机制 变成叶绿体，反之亦然，而且这种歧视 涉及线粒体中存在的特定序列，但不涉及 叶绿体转运肽。 为了验证这些假说，细胞器蛋白的基因 豌豆的合成因子(特别是EF-Tu和EF-G) 线粒体和叶绿体将被克隆和测序。 从这些密切相关的转运肽的比较中 线粒体和叶绿体多肽，序列可能 指定要预测的多肽的目的地。 线粒体和叶绿体转运肽的片段 将被删除、来回切换以及特定的序列 将通过体外定点突变而改变。正常和 诱变蛋白将通过体外合成 转录/翻译并输入到纯化的细胞器将 被研究。改变的转运肽在体外的作用， 在某些情况下，在体内，在转基因植物中，将有助于 确定转运肽中的哪些序列起作用 瞄准信号。 这些研究将有助于更好地理解 多肽是以真核生物中特定细胞器为靶标的。在……里面 从长远来看，这项工作也将增加对 细胞器的生物发生和细胞器蛋白质的合成。

The problem of organellar targeting in eukaryotic cells is addressed by studying the sorting of closely related, cytoplasmically synthesized polypeptides to mitochondria and chloroplasts in Pisum sativum, the pea. The hypotheses to be tested are that there exists some sort of discriminatory mechanism which prevents the import of mitochondrial proteins into chloroplasts, and vice versa, and that the discrimination involves specific sequences present in mitochondrial but not chloroplast transit peptides. To test these hypotheses, the genes for organellar protein synthesis factors (specifically, EF-Tu and EF-G) of pea mitochondria and chloroplasts will be cloned and sequenced. From a comparison of the transit peptides of these closely related mitochondrial and chloroplast polypeptides, sequences which may specify the destination of the polypeptide will be predicted. Segments of the mitochondrial and chloroplast transit peptides will be deleted, switched back and forth, and specific sequences will be altered by site-directed mutagenesis in vitro. Normal and mutagenized proteins will be synthesized by in vitro transcription/translation and import into purified organelles will be studied. The effects of the altered transit peptides in vitro, and in some cases, in vivo, in transgenic plants, will serve to establish which sequences within the transit peptides act as targeting signals. These studies will lead to an improved understanding of how polypeptides are targeted to specific organelles in eukaryotes. In the long term, this work will also add to the general knowledge of organellar biogenesis and of organellar protein synthesis.