Intracellular Trafficking of Plant Peroxisomal Enzymes

植物过氧化物酶体的细胞内运输

• 批准号: 9305395
• 负责人: Richard Trelease
• 金额: $ 32.24万
• 依托单位: Arizona State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9305395&HistoricalAwards=false
• 关键词: Intracellular; Trafficking; Plant; Peroxisomal; Enzymes

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.

提出了三个具体目标。第一个是建立体内植物细胞导入系统，用于实验确定植物过氧化酶体蛋白上必要的和足够的C末端靶向信号。初步数据表明，带有S-K-L-羧基的GUS基因被生物地传递到烟草细胞中，针对的是可能的过氧化物体。组织化学和双重间接免疫荧光染色表明，该系统将是值得研究的。第二个目的是发现植物过氧化物酶体蛋白上的C-末端三肽对于体内输入到培养的哺乳动物细胞中的过氧化物体是否必要和/或充分。在初步研究中，棉籽异柠檬酸裂解酶的C端三肽A-R-M被发现是进入CV-1猴肾脏和CHO细胞所必需的。第三个目标是发现将过氧化氢酶导入植物和哺乳动物细胞的过氧化物体所必需的和充分的靶向信号(S)。对于所有过氧化物体的基本构成酶--真核细胞中的过氧化氢酶，目前还没有明确的靶向信号(S)。通过意想不到的初步结果，发现了一种方法，可以区分实验进口的天然过氧化氢酶和内源性过氧化氢酶。这一突破将有助于确定C-末端结构域或N-末端结构域中的内部信号(S)是否将过氧化氢酶直接作用于真核生物的过氧化物体。细胞器内酶反应和/或整个代谢途径的区隔对于所有真核细胞的细胞过程和功能的整体协调是必不可少的。了解每个细胞器的生物发生、分化和功能(S)是必要的，然后我们才能理解细胞的复杂、完整的功能。与细胞调节和功能直接相关的细胞生物学研究中最活跃的领域之一是细胞内蛋白质向细胞器的运输。这项拟议研究的总体目标是发现和阐明针对植物细胞过氧化物体并转移到细胞内的酶上的分子靶向信号。过氧化体几乎存在于所有真核细胞中，并执行与所在细胞类型相关的各种基本代谢功能(S)。最近对过氧化物酶体靶向信号的实验研究表明，C-末端三肽基序是哺乳动物和酵母过氧化物酶体导入的必要条件和充分条件。对已发表的DNA序列的研究和用一个“共识”三肽(S-K-L)的抗血清进行的免疫印迹筛选表明，该基序在许多真核生物中普遍存在于大多数(但显然不是全部)过氧化酶体酶中。也有证据表明存在一些内部和N末端可切割的过氧化物体信号。没有针对任何天然植物过氧化物体酶的靶向信号的实验数据的一个主要原因是，无论是在体外还是在体内，都没有开发出针对植物细胞的导入系统。该项目的目的是通过开发和开发一种可在体内实验操作的植物过氧化物体进口检测系统来缩小这一信息差距。