MECHANISM OF PROTEIN TRANSPORT INTO CHLOROPLASTS

这项研究的长期目标是深入了解 将蛋白质从细胞质选择性转运到细胞器 具有特殊的代谢功能。 这项工作将侧重于 将蛋白质输入叶绿体。 叶绿体是一种 真核生物中两种产生能量的细胞器。 像 它的大多数蛋白质都是由核蛋白编码的。 基因组，作为多肽前体合成， 膜屏障，并进行蛋白水解处理。 这些研究 将有助于理解用于 在细胞中正确地传递蛋白质。 捕光叶绿素a/B结合的输入途径 蛋白质（LHCP）将被研究。 LHCP基因已经被 插入到转录载体中以产生RNA， 随后，体外标记LHCP前体（pLHCP）。 将使用重组DNA对pLHCP基因进行修饰 在一级结构中生成相应更改的方法 的先驱。 这种方法提供了一种获得 突变体的叶绿体组装尚未实现的常规 遗传学 在无细胞系统中，重组蛋白质将 合成并与分离的叶绿体一起孵育以确定 这些变化如何影响与细胞器包膜的结合， 跨膜运输，细胞器内路由， 特别感兴趣的是前体加工。 一种可溶性处理方法， 已鉴定出切割pLHCP的酶， 提纯 加工反应将被优化， 将确定pLHCP切割的决定因素。 酶 将通过柱色谱法纯化至均质， 已经开始了，来建立它的分子 组成和特异性。 编码这种酶的基因将 从互补DNA文库中分离并测序以研究 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重要作用; 它们负责释放成熟的功能性蛋白质 在细胞器中。 这项研究将与 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.