GENETICS OF FLAVIN DEHYDROGENASE-MEMBRANE INTERACTIONS

本研究的目的是确定黄素脱氢酶如何 与沙门氏菌中的膜结合电子传递链相互作用 鼠伤寒杆菌。 这项工作将重点关注两个主题： 1. put操纵子的调节。 put操纵子具有独特的控制 该机制似乎是由 putA 基因产物脯氨酸介导的 氧化酶。 当外源脯氨酸可用时，脯氨酸氧化酶与 膜，发挥黄素脱氢酶的作用；但在没有 脯氨酸，它积聚在细胞质中，充当自体 阻遏物。 确定脯氨酸氧化酶如何控制其自身表达可能 帮助我们了解其他膜结合调节蛋白的工作原理。 在 此外，putA突变的调节特性提供了遗传 膜相互作用突变体的选择。 2.黄素脱氢酶-膜相互作用。 黄素脱氢酶必须 与特定的膜位点结合，以便与 电子传输链。 然而，尚不清楚是什么成分 它们直接与电子传输链相互作用，甚至如何相互作用 它们与膜相互作用。 相互作用的遗传学研究 具有电子传递链的黄素脱氢酶将提供 理解蛋白质如何与细胞膜相互作用的新方法 体内。 涉及的所有组件以及特定的交互 required 可以通过隔离和表征两个独特的类来定义 脱氢酶突变：阻止脱氢酶发生的突变 与膜相互作用，但不消除膜的酶活性 体外脱氢酶，以及随脱氢酶改变的突变 它仍然可以结合膜，但不能与膜正确相互作用 电子传输链。 这些的遗传和生化分析 突变体将表明哪些膜成分直接与 黄素脱氢酶以及它们之间需要什么样的相互作用 黄素脱氢酶和膜电子传递链。 这样的 突变体应该提供对一般生物学问题的见解 外周膜蛋白与膜相互作用。

The objective of this study is to determine how flavin dehydrogenases interact with the membrane-bound electron transport chain in Salmonella typhimurium. This work will focus on two main topics: 1. Regulation of the put operon. The put operon has a unique control mechanism that seems to be mediated by the putA gene product, proline oxidase. When exogenous proline is available, proline oxidase binds to the membrane where it functions as flavin dehydrogenase; but in the absence of proline, it accumulates in the cytoplasm where it acts as an autogenous repressor. Determining how proline oxidase controls its own expression may help us understand how other membrane-bound regulatory proteins work. In addition, the regulatory properties of putA mutations provide a genetic selection for membrane interaction mutants. 2. Flavin dehydrogenase-membrane interactions. Flavin dehydrogenases must associate with specific membrane sites in order to interact properly with the electron transport chain. However, it is not known what component(s) of the electron transport chain they interact with directly or even how they interact with the membrane. Genetic studies on the interaction of flavin dehydrogenases with the electron transport chain will provide a novel approach for understanding how proteins interact with membranes in vivo. All of the components involved and the specific interactions required can be defined by isolating and characterizing two unique classes of dehydrogenase mutations: mutations that prevent the dehydrogenase from interacting with the membrane but don't eliminate enzymatic activity of the dehydrogenase in vitro, and mutations that alter with dehydrogenase so that it can still bind the membrane but can't properly interact with the electron transport chain. Genetic and biochemical analysis of these mutants will indicate what membrane component(s) directly interact with flavin dehydrogenases and what kinds of interactions are required between flavin dehydrogenases and the membrane electron transport chain. Such mutants should provide insight into the general biological problem of how peripheral membrane proteins interact with the membrane.