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.

关于分子马达是如何与它们的囊泡物质结合在一起的，人们知之甚少。我们现在已经证明，肌球蛋白Va，一种基于肌动蛋白的囊泡马达，通过与含有Rab27a和假设的Rab27a效应器的黑素亲和素的受体-蛋白质复合体相互作用，与其货物之一的黑素小体结合。Rab27a首先与黑素小体结合，然后招募亲黑素蛋白，后者又招募肌球蛋白Va。亲黑素通过其氨基末端以依赖于GTP的方式与Rab27a结合，并通过其羧基末端与肌球蛋白Va结合，从而建立这种联系。后一种相互作用类似于肌球蛋白Va与黑素小体共定位并影响其在体内分布的能力，完全依赖于外显子-F的存在，外显子-F是肌球蛋白Va尾部的一个选择性剪接的外显子。使用纯化的肌球蛋白Va、亲黑素和GFP标记的Rab27a的体外重建实验，结合TIRF显微镜观察肌球蛋白Va依赖于肌动蛋白的运动，表明Rab27a和亲黑素的复合体是形成功能性肌球蛋白Va受体的必要条件和充分条件。最后，将显性阴性版本的Rab27a引入活细胞，结合FRAP显微镜确定亲黑素和肌球蛋白Va在黑素小体上的停留时间，表明体内对Rab27a核苷酸状态的调节控制了肌球蛋白Va在黑素小体上的募集。此外，这些结果为基于肌动蛋白的马达提供了第一个细胞器马达的分子描述，说明了如何使用交替的外显子来指定货物，进一步扩大了Rab GTP酶及其效应器的功能库，并揭示了马达与细胞器结合的一种新的调节机制。 CARMIL，也被称为Acan125，是一种多结构域蛋白，最初是根据它与棘阿米巴I型肌球蛋白的Src同源3(SH3)结构域的相互作用而被发现的。在随后的研究中，下拉实验表明，该蛋白还与帽蛋白和Arp2/3复合体结合。我们现在已经获得了棘阿米巴与帽蛋白紧密相互作用的生化证据。在生化制剂中，CARMIL与两个多肽进行了广泛的共纯化，显微测序表明这两个多肽是棘阿米巴外壳蛋白的a-和b-亚基。CARMIL与CARMIL之间的络合物很容易被化学交联证明，但在pH为5.4时能被尺寸排除层析完全解离。超速离心、表面等离子激元共振和SH3结构域下拉分析表明，CARMIL封端蛋白的解离常数为~0.4 mm或更低。利用CARMIL融合蛋白，CARMIL的结合位点位于CARMIL的羧基末端，~200个残基，富含脯氨酸的结构域。最后，化学交联、分析超速离心法和旋转阴影电子显微镜表明，CARMIL是不对称的，它以二聚体平衡形式存在，结合常数为1.0×106M-1。综上所述，这些结果表明CARMIL与覆盖蛋白的亲和力很强(覆盖蛋白和CARMIL的细胞浓度分别为~1 mM和2 mM)，表明这两种活性都应该是生理相关的。