A myosin-like protein and GTPase required for gliding

描述（由申请人提供）：黄色粘球菌是一种长杆状细菌，它使用两个同时运行的不同电机在表面上滑动。这项工作的长期目标是确定滑翔的结构组件，并了解两个电机的方向在细胞反转过程中如何协调。近期目标将集中在 MgIA 和 MgIB 上，它们似乎在运动转换中发挥着重要作用，以及 AglZ 上，这是一种与 MgIA 相互作用的蛋白质，似乎是一个运动系统的结构成分。这些蛋白质是真核蛋白质的对应物，例如 Ras、GNRF 和肌球蛋白，它们控制着真核细胞中的重要调节过程。 RAS 突变将平衡转向 GTP 结合形式，导致真核细胞转化，最终形成肿瘤。同样，MgIA（一种 Ras 样 GTP 酶）的突变会影响 M. xanthus 中关键但非必需的过程。这些系统之间的相似性将被用来提供有关 GTP 酶及其效应子的生物学和进化信息。 mgIA 突变体的表型表明 MgIA 在两个运动系统之间传递信息，以便发动机同时反转方向。水解对于运动是必要的，因为影响 MgIA 残基的突变预计有利于 ON（GTP 结合）状态，从而减少或消除滑动。描述了表征 MgIA 生化特征的其他实验。一种类型的 mgIA 突变影响 MgIA 体内功能，但不影响体外活性，并暗示 GAP 蛋白的存在。描述了鉴定 GAP 同系物的实验。 mgIB（一种推定的 MgIA 释放因子）的破坏会降低运动性。 AglZ 是一种肌球蛋白样丝状形成蛋白，是其中一个运动系统所必需的。描述了用于检查体内和体外 AglZ 结构的动力学和极性的实验，例如 FRAP 分析。抗体和 GFP 标记的蛋白将用于检查 MgIA 和 AglZ 之间的体内和体外相互作用。

DESCRIPTION (provided by applicant): Myxococcus xanthus is a long, rod-shaped bacterium that glides over surfaces using two different motors that operate simultaneously. The long term objective of this work is to identify the structural components of gliding and to understand how the direction of the two motors is coordinated during cell reversal. The immediate goals will focus on MgIA and MgIB, which appear to play a major role in motor switching, and AglZ, a protein that interacts with MgIA and which appears to be a structural component for one motility system. These proteins are counterparts of eukaryotic proteins, such as Ras, GNRF, and myosin, that govern essential regulatory processes in eukaryotic cells. Mutations in RAS that shift the balance toward the GTP-bound form result in transformation of eukaryotic cells which can culminate in a tumor. Similarly, mutations in MgIA, a Ras-like GTPase, affect critical, but non-essential process in M. xanthus. The similarities between these systems will be exploited to provide biologic and evolutionary information about GTPases and their effectors. The phenotype of an mgIA mutant suggests that MgIA relays information between the two motility systems so that engines reverse direction simultaneously. Hydrolysis is necessary for movement because mutations that affect residues of MgIA predicted to favor the ON (GTP-bound) state reduce or abolish gliding. Additional experiments to characterize MgIA biochemically are described. One type of mgIA mutation affects MgIA function in vivo without affecting activity in vitro and hints of the existence of a GAP protein. Experiments to identify a GAP homolog are described. Disruption of mgIB, a putative release factor for MgIA, reduces motility. AglZ is a myosin-like, filament-forming protein that is required for one of the motility systems. Experiments, such as FRAP analysis, to examine the dynamics and polarity of the AglZ structure in vivo and in vitro are described. Antibody and GFP-tagged proteins will be used to examine the interaction between MgIA and AglZ in vivo and in vitro.