Structure And Function Of Unconventional Myosins

非常规肌球蛋白的结构和功能

• 批准号: 6541668
• 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/6541668
• 关键词: Dictyostelium; chimeric proteins; endoplasmic reticulum; guanosinetriphosphatases; immunologic assay /test; intracellular transport; laboratory mouse; melanocyte; melanosomes; molecular cloning; myosins; protein folding; protein isoforms; protein protein interaction; protein structure function; protoplasm motility; site directed mutagenesis; tissue /cell culture

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface is dependent on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a serves as an essential component of the melanosome receptor for myosin Va. We have now shown that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null(dilute) phenotype in wild type melanocyes is absoltutely dependent on the presence of Exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue melanosome distribution in dilute melanocytes is absolutely dependent on the presence of Exon F, since melanocyte myosin Va, and melanocyte myosin Va without Exon D colocalize and rescue, while melanocyte myosin Va without Exon F and brain myosin Va do not colocalize or rescue. These results imply that if myosin Va and Rab27a are a motor: receptor pair, their physical association should be Exon F-dependent. Consistent with this, Rab27a present in melanocyte detergent lysates binds to beads coated with purified, full-length, FLAG-tagged melanocyte myosin Va and melanocyte myosin Va lacking Exon D, but not to melanocyte myosin Va lacking Exon F, or to brain myosin Va. Furthermore, the preparation of lysates in the presence of GDP rather than GTP reduces the amount of Rab27a bound to melanocyte myosin Va by ~4 fold. Finally, the stable interaction of myosin Va and Rab27a appears to require at least one additional lysate-derived factor, since purified, GTP-loaded Rab27a does not bind appreciably to myosin Va-coated beads. Together, these results firmly establish that myosin Va and Rab27a function in the context of the melanosome as a motor: receptor pair, show that their interaction requires Exon F and at least one additional protein, and suggest that the recruitment of myosin Va on to the melanosome surface should be regulated by factors controlling the nucleotide state of Rab27a. The Dictyostelium CARMIL protein acts as a scaffold to link capping protein (CP) and the Arp2/3 complex to type I myosins through their SH3 domains. To further characterize this complex, we have purified Acanthamoeba CARMIL to homogeneity. Analytical ultracentrifugation, electron microscopy, and chemical crosslinking studies show that CARMIL is in a monomer: dimer equilibrium with an association constant of ~ 1uM. The most striking observation regarding the purification of CARMIL is that it copurifies extensively with CP. Complete dissociation of the two by gel filtration requires a mild chaotropic agent or low pH (5.4). Purified CP rebinds to CARMIL with a maximum stoichiometry of two CP heterodimers per CARMIL momomer, and with an affinity of ~ 100 nM. Given this affinity, and the cellular concentrations of CARMIL (~2 uM) and CP (~1 uM), CARMIL would be predicted to significantly influence barbed end capping in vivo. Recent in vitro experiments show that the CARMIL: CP complex can cap actin filaments, indicating that CARMIL does not function to simply sequester CP in an inactive pool. Rather, CARMIL and CP form a novel barbed end cap that (i)may be translocated to barbed ends by myosin I, and (ii) may, through its ability to also recruit and activate Arp2/3, allow the assembly of a new actin filament off of a capped end.

缺乏 GTPase Rab27a (ashen) 的黑素细胞无法进行肌球蛋白 Va 依赖性黑素体捕获，因为肌球蛋白与黑素体表面的关联取决于这种常驻黑素体膜蛋白的存在。对这些观察结果的一种解释是 Rab27a 是肌球蛋白 Va 黑素体受体的重要组成部分。我们现在已经证明，肌球蛋白 Va 尾部结构域定位于黑素体并在野生型黑素细胞中产生肌球蛋白 Va 无效（稀释）表型的能力绝对取决于外显子 F 的存在，外显子 F 是存在于黑素体尾部的两个选择性剪​​接的外显子之一。 肌球蛋白 Va 的黑素细胞剪接亚型，但不是脑剪接亚型。外显子 D（另一个黑素细胞特异性尾部外显子）不是必需的。同样，全长肌球蛋白 Va 与黑素体共定位并拯救稀黑素细胞中黑素体分布的能力绝对取决于外显子 F 的存在，因为黑素细胞肌球蛋白 Va 和没有外显子 D 的黑素细胞肌球蛋白 Va 共定位和拯救，而没有外显子 F 的黑素细胞肌球蛋白 Va 和脑肌球蛋白 Va 不共定位 或救援。这些结果意味着，如果肌球蛋白 Va 和 Rab27a 是运动受体对，那么它们的物理关联应该是外显子 F 依赖性的。与此一致，存在于黑素细胞去污剂裂解物中的 Rab27a 与涂有纯化、全长、FLAG 标记的黑素细胞肌球蛋白 Va 和缺乏外显子 D 的黑素细胞肌球蛋白 Va 的珠子结合，但不与缺乏外显子 F 的黑素细胞肌球蛋白 Va 或脑肌球蛋白 Va 结合。此外，在 GDP 存在下制备裂解物，而不是在 GDP 存在下制备裂解物。 GTP 可使与黑素细胞肌球蛋白 Va 结合的 Rab27a 量减少约 4 倍。最后，肌球蛋白 Va 和 Rab27a 的稳定相互作用似乎需要至少一种额外的裂解物衍生因子，因为纯化的、负载 GTP 的 Rab27a 不会明显与肌球蛋白 Va 包被的珠子结合。总之，这些结果坚定地证实了肌球蛋白 Va 和 Rab27a 在黑素体中作为运动受体对起作用，表明它们的相互作用需要外显子 F 和至少一种额外的蛋白质，并表明肌球蛋白 Va 向黑素体表面的募集应受到控制 Rab27a 核苷酸状态的因素的调节。 盘基网柄菌 CARMIL 蛋白充当支架，通过其 SH3 结构域将加帽蛋白 (CP) 和 Arp2/3 复合物与 I 型肌球蛋白连接起来。为了进一步表征该复合物，我们将棘阿米巴 CARMIL 纯化至均质。分析超速离心、电子显微镜和化学交联研究表明，CARMIL 处于单体：二聚体平衡状态，缔合常数约为 1uM。关于 CARMIL 纯化的最引人注目的观察结果是它与 CP 广泛共纯化。通过凝胶过滤将两者完全解离需要温和的离液剂或低 pH (5.4)。纯化的 CP 与 CARMIL 重新结合，每个 CARMIL 单体最多有两个 CP 异二聚体，亲和力约为 100 nM。考虑到这种亲和力以及 CARMIL (~2 uM) 和 CP (~1 uM) 的细胞浓度，预计 CARMIL 会显着影响体内倒刺封端。最近的体外实验表明，CARMIL：CP 复合物可以覆盖肌动蛋白丝，这表明 CARMIL 不能简单地将 CP 隔离在非活性池中。相反，CARMIL 和 CP 形成了一种新型的带刺末端帽，它 (i) 可以通过肌球蛋白 I 易位到带刺末端，并且 (ii) 可以通过其募集和激活 Arp2/3 的能力，允许从带帽末端组装新的肌动蛋白丝。