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.

人们对分子马达如何与其囊泡货物结合知之甚少。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 和亲黑素（假定的 Rab27a 效应子）的受体蛋白复合物相互作用，与其货物之一黑素体结合。 Rab27a 首先与黑素体结合，然后招募亲黑素，后者又招募肌球蛋白 Va。亲黑素通过其氨基末端以 GTP 依赖性方式与 Rab27a 结合，并通过其羧基末端与肌球蛋白 Va 结合，从而建立这种连接。后一种相互作用，类似于肌球蛋白 Va 与黑素体共定位并影响其体内分布的能力，完全依赖于外显子 F 的存在，外显子 F 是肌球蛋白 Va 尾部的选择性剪接外显子。使用纯化的肌球蛋白 Va、亲黑素和 GFP 标记的 Rab27a 进行体外重建实验，并结合 TIRF 显微镜观察肌球蛋白 Va 依赖性肌动蛋白运动，结果表明 Rab27a 和亲黑素的复合物对于形成功能性肌球蛋白 Va 受体是必要且充分的。最后，将显性失活版本的 Rab27a 引入活细胞，结合 FRAP 显微镜确定亲黑素和肌球蛋白 Va 在黑素体上的停留时间，表明体内 Rab27a 核苷酸状态的调节控制肌球蛋白 Va 招募到黑素体上。总之，这些结果提供了基于肌动蛋白的马达的细胞器马达的第一个分子描述，说明了如何使用交替的外显子使用来指定货物，进一步扩展了 Rab GTPases 及其效应器的功能库，并揭示了马达与细胞器关联的新调节机制。 CARMIL，也称为 Acan125，是一种多结构域蛋白，最初是根据其与来自棘阿米巴的 I 型肌球蛋白的 Src 同源 3 (SH3) 结构域的相互作用而被识别的。在随后对盘基网柄菌属 CARMIL 的研究中，下拉分析表明该蛋白质还结合加帽蛋白和 Arp2/3 复合物。我们现在已经获得了棘阿米巴 CARMIL 与加帽蛋白紧密相互作用的生化证据。在生化制剂中，CARMIL 与两种多肽广泛共纯化，微测序显示这两种多肽是棘阿米巴加帽蛋白的 a 和 b 亚基。 CARMIL 和加帽蛋白之间的复合物很容易通过化学交联来证明，并且可以通过 pH 5.4 的尺寸排阻色谱法完全解离。分析超速离心、表面等离振子共振和 SH3 结构域下拉测定表明 CARMIL 的加帽蛋白的解离常数约为 0.4 mM 或更低。使用 CARMIL 融合蛋白，加帽蛋白的结合位点位于 CARMIL 的羧基末端、约 200 个残基、富含脯氨酸的结构域内。最后，化学交联、分析超速离心和旋转阴影电子显微镜揭示了 CARMIL 是不对称的，并且它存在于单体：二聚体平衡状态，缔合常数为 1.0 x 106 M-1。总之，这些结果表明，CARMIL 通过亲和力与加帽蛋白自缔合和相互作用，考虑到蛋白质的细胞浓度（加帽蛋白和 CARMIL 分别约为 1 和 2 mM），表明这两种活性应该具有生理相关性。