Intracellular Trafficking of Plant Peroxisomal Enzymes

提出了三个具体目标。 一是发展 一种体内植物细胞导入系统， 植物上必需和足够的C-末端靶向信号 过氧化物酶体蛋白。 初步数据表明，GUS基因 与附加的S-K-L-COOH，并以生物射弹方式递送至培养的 烟草细胞中的过氧化物酶体。 组织化学 和双重间接免疫荧光染色表明， 值得我们去追寻 第二个目标是发现 植物过氧化物酶体蛋白C端三肽是否 必需和/或足以在体内输入到过氧化物酶体 培养的哺乳动物细胞 在初步研究中， 发现棉籽异柠檬酸裂解酶的三肽A-R-M必需 用于导入CV-1猴肾和CHO细胞。 第三 目的是发现必要的目标信号， 足以将过氧化氢酶输入植物过氧化物酶体， 哺乳动物细胞基本上没有数据说明 任何真核过氧化氢酶的特异性推定靶向信号， 所有过氧化物酶体的基本组成酶。 通过 出乎意料的初步结果，发现了一种方法， 可以区分实验性进口的天然过氧化氢酶 来自内源性过氧化物酶体过氧化氢酶。 这一突破将 允许确定C-或 N-末端结构域将过氧化氢酶导向真核生物过氧化物酶体。 %%% 酶反应和/或整个代谢的区室化 细胞器内的通路对整体协调至关重要 所有真核细胞的细胞过程和功能。 了解生物起源，分化，和功能（S）的每一个 细胞器是我们理解复杂性之前必不可少的 细胞的综合功能。 最活跃的领域之一， 直接涉及细胞调节的细胞生物学研究 其功能是将细胞内蛋白质运输到细胞器。 拟议研究的总体目标是发现并 阐明酶上的分子靶向信号， 转移到植物细胞过氧化物酶体中。 过氧体 发生在几乎所有的真核细胞中，并进行各种 与细胞类型相关的基本代谢功能， 过氧化物酶体存在。 过氧化物酶体的实验研究进展 靶向信号显示，C-末端三肽基序是 必要和足够的进口到哺乳动物和酵母 过氧化物酶体 已发表的DNA序列和蛋白质印迹的调查 用“共有”三肽（S-K-L）的抗血清筛选， 该基序对于大多数过氧化物酶体是共同的，但显然不是全部， 酶在各种真核生物中的作用。 证据还 存在一些内部和N-末端可裂解的过氧化物酶体 信号. 实验数据不存在的主要原因是 任何天然植物过氧化物酶体酶的靶向信号是， 尚未开发出体外或体内导入系统 for plant植物cell细胞. 该项目的目的是关闭， 通过开发和利用一种实验性的 可操作的体内植物过氧化物酶体输入测定系统。

Three specific objectives are proposed. The first is to develop an in-vivo plant cell import system for experimentally determining necessary and sufficient C-terminal targeting signals on plant peroxisome proteins. Preliminary data indicate that the GUS gene with an appended S-K-L-COOH and delivered biolistically to cultured tobacco cells, is directed to putative peroxisomes. Histochemical and double indirect immunofluorescent staining suggest this system will be worth pursuing. The second objective is to discover whether the C-terminal tripeptides on plant peroxisome proteins are necessary and/or sufficient for import in vivo into peroxisomes of cultured mammalian cells. In preliminary studies, the C-terminal tripeptide A-R-M of cottonseed isocitrate lyase was found necessary for import into CV-1 monkey kidney and CHO cells. The third objective is to discover the targeting signal(s) necessary and sufficient for import of catalases into peroxisomes of plant and mammalian cells. There are essentially no data elucidating the specific putative targeting signal(s) for any eukaryotic catalase, the basic constitutive enzyme of all peroxisomes. Through unexpected preliminary results, a means was discovered whereby it is possible to distinguish experimentally-imported, native catalase from endogenous peroxisomal catalase. This breakthrough will permit the determination of whether internal signal(s) within C- or N-terminal domains direct catalase to eukaryotic peroxisomes. %%% Compartmentation of enzyme reactions and/or entire metabolic pathways within organelles is essential to the overall coordination of cellular processes and functions of all eukaryotic cells. Understanding biogenesis, differentiation, and function(s) of each organelle is essential before we can comprehend the complex, integrated functions of cells. One of the most active areas of cell biological research which pertains directly to cell regulation and function is intracellular protein trafficking to organelles. The overall goal of the proposed research is to discover and elucidate molecular targeting signals on enzymes which are directed to, and translocated into, plant cell peroxisomes. Peroxisomes occur in virtually all eukaryotic cells and conduct a variety of essential metabolic function(s) related to the cell type in which the peroxisome resides. Recent experimental research on peroxisome targeting signals revealed that a C-terminal tripeptide motif was necessary and sufficient for import into mammalian and yeast peroxisomes. Surveys of published DNA sequences and western blot screens with antisera to a "consensus" tripeptide (S-K-L) indicated that the motif was common for most, but clearly not all, peroxisome enzymes among a variety of eukaryotic organisms. Evidence also exists for some internal and N-terminal cleavable peroxisome signals. A primary reason that experimental data do not exist for targeting signals on any native plant peroxisome enzyme is that import systems, either in vitro or in vivo, have not been developed for plant cells. The aim of this project is to close that informational gap by developing and exploiting an experimentally manipulable in vivo plant peroxisomal import assay system.