A myosin-like protein and GTPase required for gliding

滑行所需的肌球蛋白样蛋白和 GTP 酶

• 批准号: 7252568
• 负责人: PATRICIA L HARTZELL
• 金额: $ 25.97万
• 依托单位: UNIVERSITY OF IDAHO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252568
• 关键词: Affect; Affinity; Alleles; Antibodies; Binding; Biochemical; Biological Assay; C-terminal; Cells; Coiled-Coil Domain; Disruption; EEF1A1 gene; Equilibrium; Eukaryotic Cell; Filament; GTP Binding; GTPase-Activating Proteins; Genes; Genetic Suppression; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Hydrolysis; Immunofluorescence Immunologic; In Vitro; Localized; Location; Masks; Mediating; Membrane; Microscopic; Modeling; Molecular; Molecular Analysis; Monomeric GTP-Binding Proteins; Motor; Movement; Mutation; Myosin ATPase; Myxococcus xanthus; N-terminal; Names; Nature; Nonmuscle Myosin Type IIA; Peptide Elongation Factor Tu; Phenotype; Play; Process; Prokaryotic Cells; Proteins; Rate; Role; STY kinase; Shapes; Signal Transduction; Structure; Surface; System; Techniques; Testing; Two-Hybrid System Techniques; Work; Yeasts; base; cell motility; design; gliding bacteria; human EEF1A1 protein; in vivo; mutant; release factor; research study; retinal rods; social; tumor; yeast two hybrid interaction screening; yeast two hybrid system

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.

描述（由申请人提供）：黄色粘球菌是一种长杆状细菌，使用两种同时运行的不同马达在表面上滑行。这项工作的长期目标是确定滑翔的结构组成部分，并了解如何在细胞反转过程中协调两个电机的方向。近期目标将集中在MgIA和MgIB，这似乎在电机开关中发挥了重要作用，和AglZ，一种蛋白质，与MgIA相互作用，这似乎是一个动力系统的结构组成部分。这些蛋白质是真核蛋白质的对应物，例如Ras、GNRF和肌球蛋白，其控制真核细胞中的基本调控过程。RAS中的突变将平衡向GTP结合形式转移，导致真核细胞转化，最终导致肿瘤。类似地，MgIA（Ras样GT3）中的突变影响M中关键但非必需的过程。xanthus这些系统之间的相似之处将被利用，以提供生物和进化的GTP酶及其效应的信息。一个mgIA突变体的表型表明，MgIA中继两个运动系统之间的信息，使引擎同时反向。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残基的突变减少或消除滑动。描述了用于表征MgIA生物化学性质的另外的实验。一种类型的mgIA突变在体内影响MgIA功能，而不影响体外活性，并暗示GAP蛋白的存在。描述了鉴定GAP同源物的实验。破坏MgIB（MgIA的假定释放因子）可降低运动性。AglZ是一种肌球蛋白样的、促凝蛋白形成蛋白，其是运动系统之一所需的。实验，如FRAP分析，以检查体内和体外的AglZ结构的动力学和极性进行了描述。抗体和GFP标记的蛋白质将用于检查体内和体外MgIA和AglZ之间的相互作用。