Intracellular Trafficking of Plant Peroxisomal Enzymes

提出了三个具体目标。首先是开发一种体内植物细胞输入系统，用于实验确定植物过氧化物酶体蛋白上必要和充分的c端靶向信号。初步数据表明，GUS基因附加S-K-L-COOH，并通过生物学方式传递给培养的烟草细胞，被定向到假定的过氧化物酶体上。组织化学和双重间接免疫荧光染色表明该系统值得研究。第二个目标是发现植物过氧化物酶体蛋白上的c端三肽是否必要和/或充分地进入培养的哺乳动物细胞的过氧化物酶体。初步研究发现，棉籽异柠檬酸裂解酶的c端三肽A-R-M是进入CV-1猴肾和CHO细胞所必需的。第三个目标是发现过氧化氢酶进入植物和哺乳动物细胞过氧化物酶体所必需和充分的靶向信号。对于真核过氧化氢酶（所有过氧化物酶的基本组成酶），基本上没有数据阐明特定的假定靶向信号。通过意想不到的初步结果，发现了一种可以区分实验进口的天然过氧化氢酶和内源性过氧化物酶体过氧化氢酶的方法。这一突破将允许确定C端或n端结构域的内部信号是否将过氧化氢酶直接导向真核过氧化物酶体。细胞器内酶反应和/或整个代谢途径的区隔对所有真核细胞的细胞过程和功能的整体协调至关重要。在理解细胞复杂的综合功能之前，了解每个细胞器的生物发生、分化和功能是必不可少的。细胞生物学研究中最活跃的领域之一是直接涉及细胞调节和功能的细胞内蛋白质转运到细胞器。提出的研究的总体目标是发现和阐明酶的分子靶向信号，这些信号被导向并转运到植物细胞过氧化物酶体中。过氧化物酶体几乎存在于所有真核细胞中，并执行与过氧化物酶体所在细胞类型相关的各种基本代谢功能。最近对过氧化物酶体靶向信号的实验研究表明，c端三肽基序对于进入哺乳动物和酵母过氧化物酶体是必要和充分的。对已发表的DNA序列的调查和对“共识”三肽（S-K-L）的抗血清的western blot筛选表明，该基序在大多数真核生物中是共同的，但显然不是全部。一些内部和n端可切割的过氧化物酶体信号也存在证据。没有任何原生植物过氧化物酶体酶靶向信号的实验数据的主要原因是，无论是体外还是体内的导入系统，都没有针对植物细胞开发。该项目的目的是通过开发和利用一种实验可操作的体内植物过氧化物酶体进口测定系统来缩小这一信息差距。

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.