TARGETING OF ORGANELLAR PROTEIN SYNTHESIS FACTORS

真核细胞中细胞器靶向的问题是 通过研究密切相关的排序， 细胞质合成的多肽到线粒体， 豌豆中的叶绿体 假设是 测试表明，存在某种形式的歧视， 阻止线粒体蛋白质输入的机制 转化为叶绿体，反之亦然， 涉及存在于线粒体中的特定序列， 叶绿体转运肽 为了验证这些假设， 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， 线粒体和叶绿体多肽的序列，其可以 指定多肽的目的地将被预测。 线粒体和叶绿体转运肽的片段 会被删除，来回切换， 将通过体外定点诱变改变。 正常和 诱变的蛋白质将通过体外合成， 转录/翻译和导入纯化的细胞器将 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， 确定转运肽中的哪些序列作为 瞄准信号 这些研究将有助于更好地了解 多肽靶向真核生物中的特定细胞器。 在 从长远来看，这项工作还将增加对 细胞器生物发生和细胞器蛋白质合成。

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.