Structure And Function Of Unconventional Myosins

非常规肌球蛋白的结构和功能

• 批准号: 6822878
• 负责人: JOHN A HAMMER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6822878
• 关键词: Dictyostelium; guanosinetriphosphatases; intracellular transport; laboratory mouse; melanocyte; melanosomes; myosins; protein folding; protein isoforms; protein protein interaction; protein structure function

Little is known about how molecular motors bind to their vesicular cargo. We have now shown that myosin Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor-protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin Va. Melanophilin creates this link by binding to Rab27a in a GTP-dependent fashion through its amino terminus, and to myosin Va through its carboxy terminus. This latter interaction, similar to the ability of myosin Va to colocalize with melanosomes and influence their distribution in vivo, is absolutely dependent on the presence of exon-F, an alternatively spliced exon in the myosin Va tail. In vitro reconstitution experiments using purified myosin Va, melanophilin, and GFP-tagged Rab27a, coupled with TIRF microscopy to visualize myosin Va-dependent movement on actin, show that the complex of Rab27a and melanophilin is both required and sufficient to form a functional myosin Va receptor. Finally, introduction of dominant negative versions of Rab27a into living cells, coupled with FRAP microscopy to determine the residence time of melanophilin and myosin Va on melanosomes, shows that regulation of the nucleotide state of Rab27a in vivo controls the recruitment of myosin Va onto the melanosome. Togther, these results have provided the first molecular description of an organelle motor for an actin-based motor, illustrated how alternate exon usage can be used to specify cargo, further expanded the functional repertiore of Rab GTPases and their effectors, and revealed a novel regulatory mechanism for the association of motors to organelles. CARMIL, also known as Acan125, is a multi-domain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. We have now obtained biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL co-purified extensively with two polypeptides that were shown by microsequencing to be the a- and b-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical crosslinking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ~0.4 mM or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl terminal, ~200 residue, proline-rich domain of CARMIL. Finally, chemical crosslinking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer:dimer equilibrium with an association constant of 1.0 x 106 M-1. Together, these results indicate that CARMIL self associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (~1 and 2 mM for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.

关于分子电动机如何与囊泡货物结合的知之甚少。现在，我们已经表明，肌动蛋白VA是一种基于肌动蛋白的囊泡运动，它通过与含有Rab27a和黑色素蛋白的受体 - 蛋白质复合物相互作用（一种假定的RAB27A效应子）结合了其黑色素体。 Rab27a首先结合黑色素体，然后募集黑色素蛋白，进而募集肌球蛋白VA。黑色素蛋白通过其氨基末端以GTP依赖性方式与Rab27a结合，从而创建了这种联系，并通过其羧基末端与肌球蛋白VA结合。后一种相互作用类似于肌球蛋白VA与黑色素体共定位并影响它们在体内的分布的能力，这绝对取决于外显子F的存在，这是肌球蛋白VA尾部中剪接的外显子的存在。使用纯化的肌球蛋白VA，黑色素蛋白和GFP标记的RAB27A，与TIRF显微镜相结合以可视化肌动蛋白上的肌球蛋白VA依赖性运动，表明Rab27a和黑色素粒细胞的复合物既需要并且足以形成功能性肌球蛋白VA的肌球蛋白VA受体。最后，将Rab27a的主要负面版本引入活细胞中，再加上FRAP显微镜，以确定黑色素体上黑色素蛋白和肌球蛋白VA的停留时间，表明体内Rab27a的核苷酸状态的调节可以控制肌球蛋白VA上的甲状腺素VA上的梅拉素体。这些结果为基于肌动蛋白的电动机提供了细胞器电动机的第一个分子描述，说明了如何使用替代外显子使用来指定货物，进一步扩展了RAB GTPase及其效应子的功能库，并揭示了一种新型的调节机制，用于使电动机与细胞器结合。 Carmil，也称为ACAN125，是一种多域蛋白，最初是根据其与Acanthamoeba的I型肌动物的SRC同源性3（SH3）结构域相互作用而识别的。在随后对Carmil的dictyostelium的研究中，下拉测定法表明该蛋白还结合了封盖蛋白质和ARP2/3复合物。现在，我们已经获得了acanthamoeba carmil与封盖蛋白紧密相互作用的生化证据。在生化制剂中，Carmil与两个多肽共纯化，这些多肽通过微钉测序显示为Acanthamoeba封盖蛋白的A-和B-亚基。 Carmil和Capping蛋白之间的复合物很容易通过化学交联证明，可以通过pH 5.4处的尺寸排除色谱法完全解离。分析性超速离心，表面等离子体共振和SH3结构域下拉测定法表明，卡米尔封盖蛋白的离解常数约为0.4 mm或更低。使用卡米尔融合蛋白，显示用于封闭蛋白的结合位点显示在羧基末端，约200个残基，富含脯氨酸的卡米尔域。最后，化学交联，分析超速离心和旋转阴影电子显微镜表明，卡米尔是不对称的，它存在于单体中：二聚体平衡，缔合常数为1.0 x 106 m-1。总之，这些结果表明，鉴于蛋白质的细胞浓度（分别用于限额蛋白质和卡米尔）的细胞浓度，Carmil自我联合并与封闭蛋白相互作用，这两种活动在生理上都应相关。