A myosin-like protein and GTPase required for gliding

描述（由申请人提供）：黄粘球菌是一种长杆状细菌，它通过同时运行的两个不同的马达在表面上滑动。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上，它们似乎在运动转换中起主要作用，以及AglZ，一种与MgIA相互作用的蛋白质，它似乎是一个运动系统的结构成分。这些蛋白质是真核蛋白的对应物，如Ras、GNRF和肌球蛋白，它们在真核细胞中控制着基本的调节过程。RAS的突变使平衡向gtp结合的形式转变，导致真核细胞的转化，最终导致肿瘤。同样，MgIA（一种类似ras的GTPase）的突变也会影响黄原草的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关于gtp酶及其效应物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，从而使发动机同时反转方向。水解是运动所必需的，因为影响预测有利于ON （gtp结合）状态的MgIA残基的突变会减少或消除滑动。另外的实验，以表征MgIA生化描述。一种类型的mgIA突变影响体内mgIA功能而不影响体外活性，并提示存在GAP蛋白。描述了鉴定GAP同源物的实验。mgIB被认为是MgIA的释放因子，它的破坏会降低运动性。AglZ是一种类似肌凝蛋白的纤维形成蛋白，是一种运动系统所必需的。实验，如FRAP分析，以检查动态和极性的AglZ结构在体内和体外描述。抗体和gfp标记的蛋白将用于检测体内和体外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.