Activity and sorting of tRNA nucleotidyltransferase

蛋白质是负责细胞中几乎所有活动的生命分子。为了进行这些活动，蛋白质必须折叠成特定的三维形状，这将使它们能够与进行活动所需的物质（例如，其他蛋白质或金属离子或小分子）相互作用。此外，这些蛋白质的结构必须允许它们在细胞中稳定一段适当的时间。有些蛋白质只是短暂存在（例如，在特定条件下开启或关闭基因的蛋白质），而其他蛋白质则需要非常稳定（例如，细胞的结构成分）。此外，由于细胞不是简单的分子袋，而是被分成离散的亚细胞区室，每个区室都有特定的工作，蛋白质必须包含信号，以确保它们被分配到细胞中需要它们活动的适当区室。最终，细胞中任何蛋白质的功能都是由四个因素共同决定的：定位、活性、结构和稳定性。蛋白质必须在正确的位置（定位）具有正确的活性，在正确的时间长度（稳定性）具有适当的结构（与辅助因子相互作用）。我们使用tRNA核苷酸转移酶（一种对细胞生命至关重要的蛋白质）来探索这些因素在蛋白质功能中的相互作用。这种酶负责产生蛋白质合成所需的功能性转移rna （trna）。如果我们缺乏tRNA核苷酸转移酶，我们缺乏功能性tRNA，我们就不能制造蛋白质，我们就会死亡。有趣的是，细胞中发生蛋白质合成或tRNA成熟的所有区室（细胞质、细胞核、线粒体以及植物中的质体）都需要tRNA核苷酸转移酶。因此，这种蛋白质必须以这样一种方式折叠，不仅是一种稳定的、有活性的蛋白质，而且是一种可以被引导到多个细胞目的地的蛋白质。为了探索定位、活性、结构和稳定性如何相互作用来定义该蛋白的功能，我们使用酵母和植物tRNA核苷酸转移酶作为模型。我们对这些蛋白质进行特定的改变，并结合使用生化、生物物理、遗传和细胞生物学技术来确定这些变化如何影响定位、活性、结构和稳定性。有趣的是，我们已经表明，特定的变化可能会影响这些参数中的一个以上。例如，我们能够改变植物tRNA核苷酸转移酶，改变其定位（减少线粒体和增加质体定位），活性（降低活性到检测水平以下）和稳定性（改变热变性），但不改变其结构（通过圆二色性和荧光光谱测量）。现在我们将探讨本地化、活动和稳定性之间的相互作用。我们还将使用多种方法来鉴定细胞中与tRNA核苷酸转移酶相互作用的其他蛋白质，以了解这些其他蛋白质如何调节或控制tRNA核苷酸转移酶靶向。这些研究将使我们更好地了解蛋白质设计的多少因素结合在一起，以确保在多个细胞区室中发挥准确的功能。随着了解的增加，我们可能能够更好地设计细胞，以优化功能蛋白的递送或纠正定位错误。

Proteins are the molecules of life that are responsible for almost all of the activities occurring in cells. To carry out these activities, proteins must fold up into specific three dimensional shapes which will allow them to interact with the things that they need to carry out their activities (e.g., other proteins or metal ions or small molecules). In addition, the structures of these proteins must allow them to be stable in the cell for an appropriate length of time. Some proteins exist only transiently (e.g., those turning on or off genes under specific conditions) while others need to be very stable (e.g., structural components of the cell). Moreover, as cells are not simply bags of molecules, but are divided into discrete sub-cellular compartments each with specific jobs, the proteins must contain signals that ensure their distribution to the appropriate compartments in the cell where their activities are needed. Ultimately, the function of any protein in a cell is defined by a combination of four factors: localization, activity, structure and stability. The protein must be in the right place (localization) with the right activity, for the right length of time (stability) with the appropriate structure (to interact with accessory factors). We use tRNA nucleotidyltransferase, a protein that is essential for cell life, to explore the interplay of these factors in protein function. This enzyme is responsible for generating functional transfer RNAs (tRNAs) which are needed for protein synthesis. If we lack tRNA nucleotidyltransferase, we lack functional tRNAs, we cannot make proteins and we die. Interestingly, tRNA nucleotidyltransferase is required in all of the compartments in the cell where protein synthesis or tRNA maturation takes place (cytosol, nucleus, mitochondrion and, in plants, plastid). So, this protein must fold up in such a way as to make not only a stable, active protein but also one that can be directed to multiple cellular destinations. To explore how localization, activity, structure and stability interact to define the function of this protein, we use the yeast and plant tRNA nucleotidyltransferases as models. We make specific changes in these proteins and use a combination of biochemical, biophysical, genetic and cell biological techniques to determine how those changes affect localization, activity, structure and stability. Interestingly, we have shown that specific changes may affect more than one of these parameters. For example, we were able to make a change in a plant tRNA nucleotidyltransferase that altered its localization (reduced mitochondrial and increased plastid localization), activity (reduced activity to below the level of detection) and stability (altered thermal denaturation), but not its structure (as measured by circular dichroism and fluorescence spectroscopy). Now we will explore the interplay between localization, activity and stability. We will also use a combination of approaches to identify other proteins in the cell that interact with tRNA nucleotidyltransferase to see how these other proteins may regulate or control tRNA nucleotidyltransferase targeting. These studies will allow us to develop a better understanding of how many factors of protein design combine to ensure accurate function in multiple cellular compartments. With this increased understanding we may be able to better design cells to optimize the delivery of functional proteins or to correct errors in localization.