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状货物结合，我们所知甚少。我们现在已经证明，肌动蛋白为基础的囊泡马达，通过与含有Rab27a和嗜黑素（一种假定的Rab27a效应器）的受体蛋白复合物相互作用，将肌动蛋白Va与其货物之一黑素小体结合。Rab27a首先与黑素小体结合，然后招募亲黑素，后者又招募肌凝蛋白Va。亲黑素通过其氨基端以gtp依赖的方式与Rab27a结合，并通过其羧基端与肌凝蛋白Va结合，从而建立这种联系。后一种相互作用，类似于肌凝蛋白Va与黑素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，这是肌凝蛋白Va尾部的一个选择性剪接的外显子。体外重构实验使用纯化的肌凝蛋白Va、嗜黑素和gfp标记的Rab27a，结合TIRF显微镜观察肌动蛋白上肌凝蛋白Va依赖的运动，结果表明Rab27a和嗜黑素复合物是形成功能性肌凝蛋白Va受体所必需和充分的。最后，将Rab27a的显性阴性版本引入活细胞，结合FRAP显微镜测定亲黑素和肌凝蛋白Va在黑素小体上的停留时间，结果表明，体内对Rab27a核苷酸状态的调节控制了肌凝蛋白Va在黑素小体上的募集。总之，这些结果提供了基于肌动蛋白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外显子来指定货物，进一步扩展了Rab GTPases及其效应物的功能库，并揭示了马达与细胞器关联的一种新的调节机制。