Structure And Function Of Unconventional Myosins

缺乏GTP酶Rab27 a（灰白色）的黑素细胞在肌球蛋白Va依赖性黑素小体捕获中是无效的，因为肌球蛋白与黑素小体表面的缔合依赖于这种常驻黑素小体膜蛋白的存在。对这些观察结果的一种解释是Rab 27 a是肌球蛋白Va黑素体受体的重要成分。我们现在已经表明，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）表型的能力绝对依赖于外显子F的存在，外显子F是肌球蛋白Va的黑素细胞剪接同种型而不是脑剪接同种型的尾部中存在的两个可变剪接外显子之一。外显子D，另一个黑素细胞特异性尾外显子，是不需要的。类似地，全长肌球蛋白Va与黑素体共定位和拯救稀释的黑素细胞中的黑素体分布的能力绝对依赖于外显子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不共定位或拯救。这些结果意味着，如果肌球蛋白Va和Rab27a是一个电机：受体对，他们的物理协会应该是外显子F依赖。与此相一致，存在于黑素细胞去污剂裂解物中的Rab27 a结合至涂覆有纯化的全长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的珠粒，但不结合至缺乏外显子F的黑素细胞肌球蛋白Va或脑肌球蛋白Va。此外，在GDP而不是GTP存在下制备裂解物使结合至黑素细胞肌球蛋白Va的Rab27 a的量减少约4倍。最后，稳定的相互作用的肌球蛋白Va和Rab27a似乎需要至少一个额外的裂解物衍生的因子，因为纯化的，GTP负载Rab27a不明显结合到肌球蛋白Va包被的珠。总之，这些结果坚定地建立，肌球蛋白Va和Rab27a的功能的背景下，黑素体作为一个电机：受体对，表明它们的相互作用需要外显子F和至少一个额外的蛋白质，并建议，招聘的肌球蛋白Va的黑素体表面应受控制的Rab27a的核苷酸状态的因素。 网骨藻CARMIL蛋白充当支架以通过它们的SH3结构域将加帽蛋白（CP）和Arp 2/3复合物连接至I型肌球蛋白。为了进一步表征这种复合物，我们将阿米巴CARMIL纯化至均一。分析超离心、电子显微镜和化学交联研究表明，CARMIL处于单体：二聚体平衡，缔合常数约为1 μ M。关于CARMIL的纯化的最显著的观察是它与CP广泛共纯化。通过凝胶过滤使两者完全解离需要温和的离液剂或低pH（5.4）。纯化的CP以每个CARMIL单体两个CP异二聚体的最大化学计量重新结合CARMIL，亲和力约为100 nM。考虑到这种亲和力以及CARMIL（~2uM）和CP（~1uM）的细胞浓度，预测CARMIL将显著影响体内倒刺封端。最近的体外实验表明，CARMIL：CP复合物可以帽肌动蛋白丝，表明CARMIL不起作用，简单地隔离CP在一个不活跃的池。相反，CARMIL和CP形成了一个新的倒刺端帽，（i）可以通过肌球蛋白I移位到倒刺端，（ii）可以通过其募集和激活Arp 2/3的能力，允许新的肌动蛋白丝从加帽端组装。

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.