MECHANISM OF PROTEIN TRANSPORT INTO CHLOROPLASTS

这项研究的长期目标是深入了解 蛋白质从细胞质到细胞器的选择性运输 具有专门的代谢功能。这项工作将集中在 将蛋白质输入叶绿体。叶绿体是一种 真核生物中产生能量的两个细胞器。就像 线粒体，它的大部分蛋白质是由核编码的 基因组，作为多肽前体合成，运输到 一种膜屏障，并经蛋白质降解处理。这些研究 将导致对以下一般原则的理解 在细胞中正确地传递蛋白质。 捕光作用下叶绿素a/b结合的输入途径 将对蛋白质(LHCP)进行研究。LHCP基因已经被 插入到转录载体中以产生RNA和 随后，体外标记LHCP前体(PLHCP)。 将使用重组DNA对pLHCP基因进行修改 方法在主结构中生成相应的更改 先驱物的。这种方法提供了一种获得 叶绿体组装中尚未通过常规方法实现的突变体 遗传学。在一个无细胞的系统中，重组的蛋白质将 合成并与分离的叶绿体孵育以确定 这些变化如何影响与细胞器包膜的结合， 跨膜运输、细胞器内路径和 特别感兴趣的是前驱体加工。一种可溶加工 裂解pLHCP的酶已被鉴定并部分 纯净的。加工反应将得到优化， 将确定pLHCP切割的决定因素。这种酶 将通过柱层析法纯化至均一， 已经开始了，以建立其分子 成分和特异度。这种酶的编码基因将是 从互补DNA文库中分离并测序研究 多肽的结构及其生物合成途径。 加工酶在细胞器的生物发生中起着至关重要的作用； 它们负责释放成熟的、有功能的蛋白质 在细胞器内。这项研究将对 纠正代谢紊乱。

The long range goal of this research is to gain insight into the selective transport of proteins from the cytoplasm into organelles with specialized metabolic functions. This work will focus on the import of proteins into the chloroplast. The chloroplast is one of the two energy producing organelles in eucaryotes. Like the mitochondrion, most of its proteins are encoded by the nuclear genome, synthesized as polypeptide precursors, transported across a membrane barrier, and proteolytically processed. These studies will lead to an understanding of the general principles used to correctly route proteins in the cell. The import pathway of the light-harvesting chlorophyll a/b binding protein (LHCP) will be investigated. The LHCP gene has been inserted into a transcription vector to generate RNA and subsequently, labeled LHCP precursor (pLHCP) in vitro. Modifications in the pLHCP gene will be made using recombinant DNA methods to generate corresponding changes in the primary structure of the precursor. This approach provides a means of obtaining mutants in chloroplast assembly not yet achieved by conventional genetics. In a cell-free system, restructured proteins will be synthesized and incubated with isolated chloroplasts to determine how these changes affect binding to the organelle's envelope, transport across the membrane, intraorganelle routing and, of particular interest, precursor processing. A soluble processing enzyme that cleaves pLHCP has been identified and partially purified. The processing reaction will be optimized, and the determinants for pLHCP cleavage will be determined. The enzyme will be purified to homogeneity by column chromatographic methods, which have already been started, to establish its molecular composition and specificity. A gene coding for the enzyme will be isolated from a complementary DNA library and sequenced to study the structure of the polypeptide and its biosynthetic pathway. Processing enzymes play an essential role in organelle biogenesis; they are responsible for releasing the mature, functional proteins within the organelle. This research will have relevance to correcting metabolic disorders.