Regulation of organelle function by spatiotemporal control of gene expression

描述(申请人提供)：真核细胞的一个标志是其组织成细胞器，即空间隔间，内有执行细胞关键生化反应所需的特殊化学环境。不同细胞器的阵列需要非常不同的内部组件。受调控的蛋白质进入细胞器是特定细胞器的一个决定性性质，因此是健康细胞功能的重要一步。因此，进口机械基因表达和细胞器生物发生的过程必须在空间和时间上协调。这种协调的失败可能会对细胞造成严重的损害：许多进口的机械蛋白已知具有一种内在的能力，可以异常地插入错误的细胞器膜中。这一观察回避了以下问题：输入机械基因的表达是如何在时空上定量地与单个细胞内的细胞器生物发生相协调的？为了回答这些问题，我们转向酿酒酵母的过氧化物酶体作为模型系统。过氧化酶体是酵母中脂肪酸代谢的中心。有几个关键的性质使过氧化体导入机制(PIM)成为一个有吸引力的模型系统来回答我们主要的问题，但也许最有趣的是，许多PIM mRNAs在富含脂肪酸的细胞培养中显示出对过氧化酶体的强烈定位。这表明，通过翻译与细胞器的空间联系，mRNA的定位可能在协调PIM基因表达与过氧化体的过程中发挥重要作用。下面提出的实验结果将首次量化。 阐明基因表达的空间控制在细胞器生物发生、受调控的蛋白质输入和功能中的一个决定性方面的作用。我们的具体目标是围绕以下问题来组织的：将PIM mRNAs定位到过氧化酶体的功能作用是什么？我们将在每个目标中检验的总体假设是，PIM mRNAs的定位协调了基因表达与过氧化物酶体的生物发生，以防止编码蛋白错误定位到非过氧化体膜上。在具体目标1中，我们详细描述了脂肪酸油酸诱导过氧化体功能后PIM基因表达的时空动态的广泛的、定量的特征。在《特定目标2》中我们测试 无论是对特定目的1中研究的转录本进行特定的错位定位，都会降低PIM蛋白靶向过氧化酶体的效率或精确度。这一目标的结果将使我们能够推断PIM蛋白靶向过氧体的精确度在多大程度上是PIM mRNA与过氧体表面空间接近的结果。在特定的目标3中，我们确定了负责将Peroxin mRNAs运输到Peroxisome的组件是否在翻译开始后作用于mRNA上的顺式序列元件或反式序列元件。

DESCRIPTION (provided by applicant): A hallmark of the eukaryotic cell is its organization into organelles, spatial compartments that house specialized chemical environments needed to carry out critical biochemical reactions for the cell. The array of different organelles requires vastly different internal components. Regulated protein import into organelles is a defining property of a given organelle, and is thus a major step in the functioning of a healthy cell. The process of import machinery gene expression and organelle biogenesis must therefore be coordinated in space and time. Failure of this coordination can be severely detrimental to the cell: many import machinery proteins are known to have an intrinsic ability to aberrantly insert into incorrect organelle membranes. This observation begs the following questions: how is import machinery gene expression spatiotemporally quantitatively coordinated with organelle biogenesis within individual cells? To answer these questions, we turn to the peroxisome of Saccharomyces cerevisiae as a model system. The peroxisome is central for fatty acid metabolism in yeast. There are several key properties that render the peroxisome import machinery (PIM) an attractive model system to answer our overarching question, but perhaps the most intriguing is that many PIM mRNAs exhibit strong localization to the peroxisome upon culture of cells in fatty acid rich media. This suggests that mRNA localization might play a major role in coordinating PIM gene expression with peroxisomes by spatially linking translation with the organelle. The results of the experiments proposed below will for the first time quantitatively illuminate the role of spatial control of gene expression in a defining aspect of organelle biogenesis, regulated protein import, and thus function. Our Specific Aims are organized around addressing the following question: what is the functional role of localizing PIM mRNAs to the peroxisome? The overall hypothesis we will test in each Aim is that localization of the PIM mRNAs coordinates gene expression with peroxisome biogenesis in order to prevent mislocalization of the encoded proteins to non-peroxisomal membranes. In Specific Aim 1 we detail a broad, quantitative characterization of spatiotemporal PIM gene expression dynamics following induction of peroxisomal function by the fatty acid oleic acid. In Specific Aim 2 we test whether specifically mislocalizing the transcripts studied in Specific Aim 1 decreases the efficiency or precision of PIM protein targeting to the peroxisome. The results from this Aim will allow us to conclude how much of the precision of PIM protein targeting to peroxisomes is a consequence of spatial proximity of the PIM mRNA to the peroxisomal surface. In Specific Aim 3 we determine whether the components responsible for trafficking peroxin mRNAs to the peroxisome act on cis sequence elements on the mRNA or in trans following initiation of translation.