Regulation of organelle function by spatiotemporal control of gene expression

描述（由申请人提供）：真核细胞的一个标志是其组织成细胞器，空间隔室容纳细胞进行关键生化反应所需的专门化学环境。不同细胞器的阵列需要非常不同的内部组件。调节蛋白质进入细胞器是给定细胞器的定义属性，因此是健康细胞功能的重要步骤。因此，输入机器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对细胞严重有害：已知许多输入机器蛋白具有异常插入不正确的细胞器膜的内在能力。这一观察回避了以下问题：如何进口机械基因表达时空定量协调与细胞器的生物合成在个别细胞？为了回答这些问题，我们转向过氧化物酶体的酿酒酵母作为一个模型系统。过氧化物酶体是酵母中脂肪酸代谢的中心。有几个关键的属性，使过氧化物酶体输入机器（PIM）一个有吸引力的模型系统，以回答我们的首要问题，但也许最有趣的是，许多PIM mRNA表现出强烈的本地化的过氧化物酶体培养细胞在富含脂肪酸的培养基。这表明，mRNA定位可能发挥了重要作用，协调PIM基因表达与过氧化物酶体的空间连接翻译与细胞器。下面提出的实验结果将首次定量地 阐明基因表达的空间控制在细胞器生物发生，调节蛋白质进口，从而功能的定义方面的作用。我们的具体目标是围绕解决以下问题：定位PIM mRNA的过氧化物酶体的功能作用是什么？我们将在每个目标中测试的总体假设是PIM mRNA的定位协调基因表达与过氧化物酶体生物发生，以防止编码的蛋白质错误定位到非过氧化物酶体膜。在具体目标1，我们详细介绍了广泛的，定量表征的时空PIM基因表达动态诱导过氧化物酶体功能的脂肪酸油酸。在具体目标2中，我们测试 是否特异性地错误定位在特异性目标1中研究的转录物降低了PIM蛋白靶向过氧化物酶体的效率或精确度。该目的的结果将使我们能够得出结论，PIM蛋白靶向过氧化物酶体的精确度是PIM mRNA与过氧化物酶体表面空间接近的结果。在具体目标3中，我们确定负责运输过氧化物酶mRNA到过氧化物酶体的组分是否作用于mRNA上的顺式序列元件或翻译起始后的反式。

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.