PROTEIN METHYLATION IN PHOTOTAXIS AND CHEMOTAXIS

我们将研究蛋白质甲基化与受体的关系 光敏和化学传感信号转导中的激发 嗜盐杆菌的代谢途径。研究的一个主要发现是 细菌趋化性是整膜的可逆甲基化 “信号转导”蛋白在适应 趋化性刺激。最近，一种使用视紫红质样体的趋光性系统 光感受器(慢循环细菌视紫红质，“SR”)已被发现 控制盐生梭子藻的兴奋和适应运动反应 类似于细菌的趋化性。Sr蛋白经历了两次 不同的光化学反应周期，取决于激发 波长。一个周期产生吸引信号，而另一个周期产生吸引信号 会产生排斥信号。这些信号的产生可以是 通过控制激励的光谱组成来控制的 灯。几个实验室的初步研究表明蛋白质 甲基化反应参与锶介导的趋光性以及 在这些细胞中的趋化行为。我们的目标是：(1) 描述H。 盐生细菌趋光性；(2)评估它们与光激发的关系。 两个锶的光化学反应循环到甲基化的激活 系统。 蛋白质甲基化将通过体内标记(~3H)甲基进行监测， 用聚丙烯酰胺凝胶电泳法和放射标记法分离蛋白质 用荧光自显影和闪烁计数进行检测。在相同的情况下 其中光刺激诱导的甲基化是 在监测下，锶的光化学反应将通过 时间分辨闪光光解。我们的理由是，这一组合 详细的受体信息和蛋白质甲基化数据将澄清 受体兴奋与甲基化/去甲基化的关系 反应。利用光控制sr信号的能力将允许我们 探讨趋光性甲基化系统对 时间分辨率和可控性很难获得的刺激 化学梯度对趋化系统的刺激作用。

We will study the relationship of protein methylation to receptor excitation in the photosensory and chemosensory signal transduction pathways of Halobacterium halobium. A major finding from studies of bacterial chemotaxis is that reversible methylation of integral membrane "signal transducer" proteins plays a critical role in adapatation to chemotaxis stimuli. Recently a phototaxis system using a rhodopsin-like photoreceptor (the slow-cycling bacterial rhodopsin, "SR") has been shown to govern excitation and adaptation motility responses in H. halobium analogous to those of bacterial chemotaxis. The sR protein undergoes two different photochemical reaction cycles, depending on excitation wavelength. One cycle generates an attractant signal, and the other generates a repellent signal. The generation of these signals can be controlled by controlling the spectral composition of the excitation light. Initial work from several laboratories indicates protein methylation reactions participate in the sR-mediated phototaxis as well as in chemotaxis behavior in these cells. Our objectives are: (1) to characterize the components of the protein methylation system in H. halobium phototaxis; (2) to assess their relationship of photoexcitation of the two sR photochemical reaction cycles to activation of the methylation system. Protein methylation will be monitored by in vivo labeling with (3H)methyl, protein separation by polyacrylamide gel electrophoresis, and radiolabel detection by autofluorgraphy and scintillation counting. In the same in vivo preparations in which the photostimulus-induced methylation is monitored, the photochemical reactions of sR will be analyzed by time-resolved flash photolysis. Our rationale is tht this combination of detailed receptor information and protein methylation data will clarify the relationship of receptor excitation to the methylation/demethylation reactions. The ability to use light to control sR signaling will permit us to probe some properties of the phototaxis methylation system's response to stimuli with a time resolution and control difficult to obtain with stimulation of chemotaxis systems by chemical gradients.