Regulation of Cellular Growth and Division by Ubiquitin-Mediated Proteolysis

细胞分裂周期由定时合成和随后的泛素介导的蛋白分解来协调。 关键的调控蛋白。已知的调节细胞周期的两种泛素连接酶复合体是 后期促进复合体(APC)和Skp1-Cul1-F-box复合体(SCF)。然而，有很多 细胞周期调节因子，其周转机制尚不清楚，以及大量的泛素 目标尚未确定的连接酶。为了更好地了解泛素连接酶如何控制细胞 循环中，我们开发了一种方法，该方法利用表达GFP融合蛋白的菌株库和高通量 显微镜来鉴定酵母中的泛素连接酶靶标，并使用这种方法来鉴定 泛素连接酶以73个不稳定的细胞周期调节因子为靶标进行破坏。在提出的实验中 在这里，我们将确定这些细胞周期蛋白由特定的E3s周转的机制，并检查 这些周转事件对正常细胞周期进程的重要性。SCF(Gn‘l)泛素连接酶是 已知以细胞周期和代谢调节因子为目标进行破坏。我们之前确定了 转录因子Tye7作为干细胞因子(Grr1)的靶标。Tye7调节代谢基因的转录，本身就是 在整个细胞周期中转录调控。我们将拘留9名Tye7的降级目标和 分析阻断SCF中介营业额的后果。最后，我们将研究细胞周期是如何 代谢因素影响SCF连接酶活性。SCF连接酶通过多种途径中的一种识别靶 称为F-box蛋白的模块化适配器亚基。目前尚不清楚有多少F-box蛋白与 以及这些复合体是否在整个细胞周期中发生变化。我们将分析云函数 在不同的细胞周期和生长状态下的复杂成分来解决这些问题。

The cell division cycle is orchestrated by the timed synthesis and subsequent ubiquitin-mediated proteolysis of key regulatory proteins. Two ubiquitin ligase complexes that are known to regulate the cell cycle are the Anaphase Promoting Complex (APC) and Skp1-Cul1-F-box (SCF) complex. However, there are numerous cell cycle regulators for which the mechanism of turnover remains unknown, and a large number of ubiquitin ligases whose targets have not been identified. To better understand how ubiquitin ligases control the cell cycle, we developed a method that utilizes a library of strains expressing GFP-fusion proteins and highthroughput microscopy to identify ubiquitin ligase targets in yeast, and are using this approach to identify the ubiquitin ligases that target 73 unstable cell cycle regulators for destruction. In the experiments proposed here, we will determine the mechanism of turnover of these cell cycle proteins by specific E3s, and examine the importance of these turnover events for nonnal cell cycle progression. The SCF(Gn'l) ubiquitin ligase is known to target both cell cycle and metabolic regulators for destruction. We previously identified the transcription factor Tye7 as a SCF(Grrl) target. Tye7 regulates transcription of metabolic genes and is itself transcriptionally regulated throughout the cell cycle. We will detennine Tye7 is targeted for degradation and analyze the consequence of blocking SCF-mediated turnover. Finally, we will investigate how the cell cycle and metabolic factors influence SCF ligase activity. SCF ligases recognize targets through one of many modular adaptor subunits called F-box proteins. It is unknown how many F-box proteins complex with the SCF at any one time, and whether these complexes change throughout the cell cycle. We will analyze SCF complex composition in different cell cycle and growth states to address these questions.