Plant Protein Farnesyltransferase: Molecular and FunctionalAnalyses

蛋白法尼基转移酶（FTase）是一种异二聚体酶，通过将异戊二烯基片段连接到c端半胱氨酸上，在翻译后修饰蛋白质。在酵母和动物中发现，这种修饰是参与细胞周期控制和分化的许多调节蛋白的膜结合和体内活性所必需的。该项目的主要重点是阐明FTase和蛋白戊烯酰化在介导植物发育过程中的作用。在最近的实验中，我们克隆并鉴定了一个编码豌豆FTase β亚基的cDNA克隆，在植物细胞中证实了蛋白烯酰化，并在豌豆幼苗和悬浮培养的番茄细胞的顶芽中分析了FTase转录水平。在这两种实验系统中，在活跃的细胞分裂开始之前，检测到FTase mRNA的快速，短暂的增加。这些结果与FTase介导植物中控制细胞分裂的信号转导通路关键组分活性的假设一致，类似于其在真菌和哺乳动物系统中控制细胞周期的关键作用。为了验证这一假设，我们将从豌豆中克隆出FTase α亚基的cDNA，并利用体外和体内瞬时表达蛋白质前置酰化底物的植物细胞来描述豌豆FTase的生化和生理特性。详细分析豌豆Ftase基因表达的时空格局，为其在植物发育中的功能意义提供线索。通过反义rna或竞争性肽抑制剂抑制Ftase表达的植物细胞和稳定转化的植物，将监测Ftase的表型、细胞和分子变化，以直接评估Ftase在调节植物细胞分裂和分化中的作用。最近发现，在某些生理条件下，某些蛋白质通过异戊烯基共价连接到特定的c端氨基酸而被修饰。异戊二烯基团是一种有机分子，在脂肪或油中比在水中更容易溶解。因此，这种修饰的作用是使原本溶解在细胞质中的蛋白质附着在细胞膜上。它们是由脂肪物质组成的。现在越来越清楚的是，特定蛋白质与膜的关联是调节细胞分裂、膜融合和各种其他细胞生物学过程的生化事件链中的一个关键事件。这种重要的调节机制已经在哺乳动物细胞和酵母中被描述和积极研究，但直到现在还不知道它在高等植物中的可能存在或作用。这是本研究的主题。这些研究人员已经确定了该酶的一部分基因，该酶在豌豆蛋白质中进行异戊二烯基的添加，并将使用分子生物学技术进一步表征该酶并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.