A comprehensive analysis of yeast Elongator phosphorylation and its functional consequences

在蛋白质分子内的特定点添加和去除磷酸基团（“蛋白质磷酸化”）是调节蛋白质功能的通用方法。磷酸基团通过称为蛋白激酶的酶添加，并通过称为蛋白磷酸酶的相反活性去除。我们感兴趣的是一种名为“延伸蛋白”的蛋白质复合体，它存在于所有高等细胞中，包括植物和动物，如我们自己。我们一直使用酵母细胞作为这些研究的模型系统，因为它们提供了一个实验系统，其中遗传和生物化学方法可以以一种特别强大的方式结合起来。Elongator已经被提出扮演许多细胞角色，但最近发现它对转移RNA的正确功能很重要，转移RNA是细胞使用其遗传模板制造更多蛋白质分子所需的特殊分子，这可能是Elongator的主要功能。我们发现，延伸复合体的一个组成部分似乎受到磷酸化的调节，尽管这可能意味着延伸复合体在所有情况下都不完全活跃。因此，细胞合成特定蛋白质的能力可以通过这种机制来控制。在我们提出的工作中，我们将联合收割机结合我们两个实验室的专业知识，研究如何以及为什么延长器可能受到磷酸化的调节。我们将研究Elongator活性的变化是否会影响细胞中特定蛋白质的合成，并确定参与磷酸化Elongator的蛋白激酶以及由这些蛋白激酶修饰的Elongator组分蛋白质中的特定位点。通过定义磷酸化的各个位点，我们可以产生根本不能被磷酸化或模拟磷酸基团存在的突变体。我们将使用这样的突变体，以建立一个详细的图片如何具体的磷酸化事件影响伸长率的功能。

The addition and removal of phosphate groups at specific points within protein molecules ('protein phosphorylation') is a universal means by which protein function can be regulated. Phosphate groups are added by enzymes termed protein kinases and removed by opposing activities termed protein phosphatases. We are interested in a complex of proteins termed 'Elongator' that is found in all higher cells including those of plants and animals such as ourselves. We have been using yeast cells as a model system for these studies because they provide an experimental system in which both genetic and biochemical approaches to biological questions can be combined in a particularly powerful way. Elongator has been proposed to play a number of cellular roles but most recently it has been discovered that it is important for the correct functioning of transfer RNAs, special molecules that are required for cells to make more protein molecules using their genetic templates, and this is probably Elongator's primary function. We have found that one of the components of the Elongator complex appears to be regulated by phosphorylation, even though this might mean that Elongator is not fully active under all circumstances. Thus the cell's ability to synthesis specific proteins may be controlled through this mechanism. In our proposed work we will combine the expertise of our two laboratories to study both how and why Elongator may be regulated by phosphorylation. We will examine whether changes in Elongator activity lead to effects on the synthesis of specific proteins in the cell and identify both the protein kinases involved in phosphorylating Elongator and the specific sites in the component proteins of Elongator that are modified by these protein kinases. By defining individual sites of phosphorylation we can generate mutants that either cannot be phosphorylated at all or that mimic the presence of a phosphate group. We will use such mutants to build up a detailed picture of how specific phosphhorylation events affect Elongator function.