Structure and function of the protein chaperone Tti2

许多蛋白质的折叠都需要伴侣。伴侣还可以解开错误折叠的蛋白质或将它们作为降解的目标。伴侣在应对压力方面尤其关键；因此，全球变暖使从基因或化学角度调节其活动的机制变得越来越有价值。我们的研究主要集中在TTT伴侣复合体上。这种复合体的组成成分TEL2、Tti1和Tti2在真核生物中是必不可少的。利用酿酒酵母的遗传选择，我们鉴定了TTI2的突变，该突变增强了Pikk(磷脂酰肌醇3-激酶相关蛋白)家族成员的折叠。这种效应是由于Tti2功能的部分丧失导致的，这导致我们假设Tti2以缓慢的折叠蛋白质为目标进行降解。TEL2和TTI1没有发现类似的突变，提示Tti2的S功能可能是独一无二的。我们研究计划的目标是确定TTT-复合体组件的功能。首先将重点放在Tti2上，因为它可能有独特的活动，而且我们有许多关键工具来分析它。我们的研究使用酵母，因为酵母允许的实验方法组合是解决基本生物学问题的理想选择。这五年的具体目标是：*1.确定TTT-复合体各组成部分的蛋白质相互作用网络。当TEL2、Tti1和Tti2的相互作用伙伴确定后，它们的功能将得到更明确的定义。我们将使用生化方法来确定这三种蛋白质的相互作用伙伴。通过突变绘制相互作用表面，然后将诱变的等位基因导入细胞，以检验新的相互作用的意义。*2.表征Tti2的结构/功能关系。利用单基因进化，我们已经确定了Tti2中不变残基的簇。这些残基将发生突变，它们的表型后果将通过我们构建的tti2基因敲除菌株中的质粒洗牌来确定。将检测突变对Tti2的表达、定位、Pikk蛋白的折叠以及蛋白质-蛋白质相互作用的影响。减少细胞生长的等位基因将是抑制基因研究的宝贵工具，这将指导实验确定Tti2的功能。*3.确定Tti2的“客户”蛋白。已经证明了TTT蛋白在Pikk家族成员折叠中的重要性。目前尚不清楚是否存在其他客户端。我们将利用条件等位基因寻找依赖于Tti2和TEL2表达的蛋白质，并在细胞培养中通过氨基酸稳定同位素标记进行蛋白质鉴定。识别靶标和相互作用蛋白的第二种方法将是通过BioID策略。这种方法是为哺乳动物细胞设计的，所以我们将从细胞培养中识别哺乳动物Tti2的靶点开始。

The folding of many proteins requires chaperones. Chaperones also untangle misfolded proteins or target them for degradation. Chaperones are particularly critical in the response to stress; thus global warming is making mechanisms to modulate their activity genetically or chemically increasingly valuable. Our studies focus on the TTT chaperone complex. The components of this complex, Tel2, Tti1 and Tti2, are essential in eukaryotes. Using genetic selections in S. cerevisiae we identified mutations in TTI2 that enhance the folding of a member of the PIKK (Phosphatidylinositol 3-kinase-related kinase) protein family. This effect results from partial loss of Tti2 function, leading us to hypothesize that Tti2 targets slowly folding proteins for degradation. Similar mutations are not found in TEL2 or TTI1, suggesting that Tti2's function may be unique. The goal of our research program is to determine the function of the components of the TTT-complex. An initial emphasis will be placed on Tti2, because of its potentially unique activities and since we have many of the key tools for its analysis. Our studies use yeast because the combination of experimental approaches yeast allow is ideal for solving fundamental biological problems. Specific objectives for this five-year period are:******1. Identify the protein interaction networks of the components of the TTT-complex. The functions of Tel2, Tti1 and Tti2 will be more clearly defined when their interacting partners are determined. We will determine the interaction partners of the three proteins using biochemical approaches. The significance of novel interactions will be examined by mapping the interaction surface through mutagenesis, then introducing the mutagenized alleles into cells.******2. Characterize the structure/function relationships of Tti2. Using unigenic evolution we have identified clusters of invariant residues in Tti2. These residues will be mutated and their phenotypic consequences determined by plasmid shuffling in a tti2 knockout strain we constructed. The effects of the mutations on Tti2 expression, localization, folding of the PIKK proteins, and protein-protein interactions will be examined. Alleles that reduce cell growth will be valuable tools for suppressor genetic studies that will guide experiments to determine the function of Tti2.******3. Identify the "client" proteins for Tti2. The importance of the TTT proteins in the folding of members of the PIKK family has been demonstrated. Whether other clients exist is unknown. We will search for proteins whose expression depends on Tti2 and Tel2 using conditional alleles and protein identification through stable isotope labeling with amino acids in cell culture. A second approach to identify both targets and interacting proteins will be through the BioID strategy. This method was designed for mammalian cells, so we will start by identifying targets of mammalian Tti2 in cell culture.