A myosin-like protein and GTPase required for gliding

描述(申请人提供)：黄色粘球菌是一种长的杆状细菌，使用两个同时运行的不同马达在表面上滑动。这项工作的长期目标是确定滑行的结构成分，并了解在细胞逆转过程中两个马达的方向是如何协调的。近期的目标将集中在mGIA和MGIB，它们似乎在电机开关中发挥主要作用，以及AglZ，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。这些蛋白质与真核细胞中的RAS、GNRF和肌球蛋白等蛋白质相对应，这些蛋白质控制着真核细胞中的基本调控过程。RAS的突变使平衡向GTP结合的形式转移，导致真核细胞的转化，最终导致肿瘤。类似地，MGIA(一种类似RAS的GTP酶)的突变会影响黄曲霉的关键但非必要的过程。这些系统之间的相似性将被用来提供关于GTP酶及其效应物的生物和进化信息。MgIA突变体的表型表明，mgIA在两个运动系统之间传递信息，从而引擎同时逆转方向。水解对于运动是必要的，因为影响mGIA残基的突变被预测为有利于打开(GTP结合)状态减少或取消滑行。描述了用于表征镁离子交换膜生物化学特性的其他实验。一种类型的mgIA突变会影响体内的mgIA功能，而不会影响体外的活性，也不会暗示存在GAP蛋白。描述了识别GAP同系物的实验。阻断MGIB，一种可能的镁释放因子，降低了运动能力。AglZ是一种肌球蛋白样丝形成蛋白，是运动系统之一所必需的。描述了实验，如FRAP分析，以检查体内和体外AglZ结构的动力学和极性。抗体和GFP标记的蛋白将被用来检测体内和体外MGIA和AglZ之间的相互作用。

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.