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.

关于分子马达如何与它们的囊泡货物结合，我们知之甚少。我们现在已经表明，肌球蛋白Va，肌动蛋白为基础的囊泡电机，结合到它的货物之一，黑素体，通过与受体蛋白复合物含有Rab 27 a和黑素，一个假定Rab 27 a效应。Rab 27 a首先与黑素体结合，然后招募亲黑素蛋白，亲黑素蛋白又招募肌球蛋白Va。亲黑素通过其氨基末端以GTP依赖性方式与Rab 27 a结合，并通过其羧基末端与肌球蛋白Va结合来产生这种连接。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在体内分布的能力，绝对依赖于外显子F的存在，外显子F是肌球蛋白Va尾中的选择性剪接外显子。在体外重建实验中使用纯化的肌球蛋白Va，亲黑素蛋白，GFP标记的Rab 27 a，再加上TIRF显微镜，以可视化肌球蛋白Va依赖的肌动蛋白的运动，表明Rab 27 a和亲黑素蛋白的复合物是必需的，足以形成一个功能性的肌球蛋白Va受体。最后，引入显性负性版本的Rab 27 a到活细胞中，再加上FRAP显微镜，以确定黑素亲和素和肌球蛋白Va在黑素体上的停留时间，表明Rab 27 a在体内的核苷酸状态的调节控制肌球蛋白Va到黑素体上的募集。总之，这些结果提供了第一个分子描述的细胞器电机肌动蛋白为基础的电机，说明如何交替外显子使用可以用来指定货物，进一步扩大了功能库的Rab GTP酶及其效应器，并揭示了一种新的调节机制，电机协会的细胞器。 CARMIL，也称为Acan 125，是一种多结构域蛋白质，最初是根据其与棘阿米巴I型肌球蛋白的Src同源性3（SH 3）结构域的相互作用而鉴定的。在随后对来自网骨藻的CARMIL的研究中，下拉测定表明该蛋白质还结合加帽蛋白和Arp 2/3复合物。我们现在已经获得了阿米巴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，所述两种多肽通过微测序显示为阿米巴加帽蛋白的a-和b-亚基。CARMIL和加帽蛋白之间的复合物，这是很容易证明的化学交联，可以完全解离的大小排阻色谱法在pH 5.4。分析性超离心、表面等离子体共振和SH 3结构域下拉测定表明，加帽蛋白对CARMIL的解离常数为约0.4 mM或更低。使用CARMIL融合蛋白，显示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，分析超离心，和旋转阴影电子显微镜显示，CARMIL是不对称的，它存在于一个单体：二聚体平衡与缔合常数为1.0 × 106 M-1。总之，这些结果表明CARMIL自身缔合并与加帽蛋白相互作用，考虑到蛋白质的细胞浓度（加帽蛋白和CARMIL分别为约1和2 mM），其亲和力表明两种活性应该是生理相关的。