Plant Protein Farnesyltransferase: Molecular and FunctionalAnalyses

蛋白法尼基转移酶（FTase）是异二聚体酶， 通过连接异戊二烯基， 在一些实施方案中，所述半胱氨酸基团与C-末端半胱氨酸连接。 这种修改，在 酵母和动物，是膜结合和体内 参与细胞周期的许多调节蛋白的活性 控制和分化。 建议的主要重点是 项目是阐明FTase和蛋白质的作用 异戊烯化在介导植物发育过程中的作用。 近几 在实验中，编码豌豆FTase β亚基的cDNA克隆具有 已被克隆和表征，蛋白质异戊二烯化已被 证明在植物细胞中，和FTase转录水平已被证实， 分析了豌豆幼苗顶芽和悬浮培养 番茄细胞 在这两个实验系统中， FTase mRNA的增加是在活动期开始前检测到的。 细胞分裂 这些结果与假设一致 FTase介导的关键成分的活动， 控制植物细胞分裂的信号转导途径， 类似于其在真菌细胞周期控制中的关键作用， 哺乳动物系统 为了验证这一假设，我们将FTase的cDNA α亚基将从豌豆中克隆出来， 豌豆FTase的生理特性将被描绘使用两个 体外测定和体内植物细胞瞬时表达 蛋白质异戊二烯化底物。 详细分析时间 豌豆脂肪酶基因表达的空间模式将被执行 为了解其在植物中的功能意义提供线索 发展 植物细胞和稳定转化的植物，抑制 反义RNA或竞争性肽在Ftase表达中的作用 抑制剂，将监测表型，细胞， 分子变化，以直接评估FTase在 调节植物细胞分裂和分化。 %%% 最近发现某些蛋白质被修饰， 在某些生理条件下，通过共价连接 异戊二烯基基团与特定的C-末端氨基酸连接。 异戊二烯基 基团是一种有机分子， 而不是在水中。 因此，这种修改具有 使蛋白质溶解的效果 附着在细胞膜上。 其由脂肪材料组成。 现在很明显 特定蛋白质与细胞膜的结合 调节细胞的生化事件链中的关键事件 分裂、膜融合和其他各种细胞生物学过程 流程. 这一重要的调节机制已经被描述为 目前正在哺乳动物细胞和酵母中进行研究， 到目前为止，还没有人知道它的存在或作用。 在高等植物中。 这是本研究的主题。 这些 研究人员已经确定了这种酶的一部分基因 将异戊二烯基添加到豌豆的蛋白质中 并将使用分子生物学技术来表征 进一步研究其在植物中的可能功能。

Protein farnesyltransferase (FTase) is a heterodimeric enzyme that post-translationally modifies proteins by attaching an isoprenyl moiety to a C-terminal cysteine. This modification, identified in yeast and animals, is required for membrane association and in vivo activities of many regulatory proteins involved in cell cycle control and differentiation. The major focus of the proposed project is to elucidate the role(s) of FTase and protein prenylation in mediating plant developmental processes. In recent experiments, a cDNA clone encoding a pea FTase beta subunit has been cloned and characterized, protein prenylation has been demonstrated in plant cells, and FTase transcript levels have been analyzed in apical buds of pea seedlings and suspension-cultured tomato cells. In both experimental systems, a rapid, transient increase in FTase mRNA was detected preceding the onset of active cell division. These results are consistent with the hypothesis that FTase mediates the activity of critical components of the signal transduction pathways controlling cell division in plants, analogous to its key role in cell cycle control in fungal and mammalian systems. To test this hypothesis, a cDNA for the FTase alpha subunit will be cloned from pea and biochemical and physiological properties of pea FTase will be delineated using both in vitro assays and in vivo plant cells transiently expressing protein prenylation substrates. Detailed analyses of the temporal and spatial patterns of pea Ftase gene expression will be performed to provide clues as to its functional significance in plant development. Plant cells and stably transformed plants, inhibited in Ftase expression by antisense RNAs or competitive peptide inhibitor, will be monitored for phenotypic, cellular, and molecular changes to directly assess the role of FTase in regulating plant cell division and differentiation. %%% It has recently been discovered that certain proteins are modified, under some physiological conditions, by covalent attachment of isoprenyl groups to a specific C-terminal amino acid. Isoprenyl groups are a type of organic molecule that tends to be more soluble in fats or oils than in water. This modification, then, has the effect of allowing proteins that would otherwise remain dissolved in the watery cytoplasm of a cell to attach to cellular membranes. which are composed of fatty materials. It is now becoming clear that the association of particular proteins with membranes is a critical event in a chain of biochemical events that regulate cell division, membrane fusion, and a variety of other cell biological processes. This important regulatory mechanism has been described and is actively being studied in mammalian cells and in yeast, but until now nothing has been known of its possible existence or role in higher plants. This is the subject of this research. These investigators have identified the gene for one part of the enzyme that carries out addition of isoprenyl groups to proteins in peas and will use techniques of molecular biology to characterize the enzyme further and to study its possible function in plants.