Structure And Function Of Unconventional Myosins

缺乏GTP酶Rab27a的黑素细胞在肌球蛋白Va依赖的黑素小体捕获中被禁用，因为肌球蛋白与黑素小体表面的联系依赖于这种驻留的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的黑素小体受体的重要组成部分。我们现在已经证明，在野生型黑色素细胞中，肌球蛋白Va尾部结构域定位于黑素小体并产生肌球蛋白Va缺失(稀释)表型的能力完全依赖于外显子F的存在，外显子F是存在于黑素细胞剪接的肌球蛋白Va亚型尾部的两个选择性剪接外显子之一，而不是脑剪接的亚型。外显子D是另一个黑素细胞特有的尾部外显子，不是必需的。同样，全长肌球蛋白Va与黑素小体共定位并挽救稀薄黑素细胞中黑素小体分布的能力完全取决于外显子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不共定位或拯救。这些结果表明，如果肌球蛋白Va和Rab27a是一对运动：受体，它们的物理联系应该是外显子F依赖的。与此一致的是，存在于黑素细胞洗涤剂裂解物中的Rab27a与包被纯化的全长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结合，但不与缺乏外显子F的黑素细胞肌球蛋白Va结合，也不与脑肌球蛋白Va结合。此外，在GDP而不是GTP存在的情况下制备的裂解物将与黑素细胞肌球蛋白Va结合的Rab27a的数量减少了~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个额外的裂解产物衍生因子，因为纯化后，负载GTP的Rab27a不会明显地结合到肌球蛋白Va包被的珠子上。综上所述，这些结果证实了肌球蛋白Va和Rab27a在黑素小体作为运动受体对的背景下的功能，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，并提示肌球蛋白Va在黑素小体表面的募集应该受到控制Rab27a核苷酸状态的因素的调节。DictyostelialCARMIL蛋白作为骨架，通过其SH3结构域将Cp和Arp2/3复合体与I型肌球蛋白连接起来。为了进一步鉴定这个复合体，我们将棘阿米巴纯化成均一种。分析超速离心、电子显微镜和化学交联研究表明，CARMIL处于二聚体平衡状态，结合常数约为1微米。关于CARMIL的纯化，最引人注目的观察是它与CP广泛交配。通过凝胶过滤将两者完全解离需要温和的变色剂或低pH值(5.4)。纯化的CP与CARMIL重新结合，每个CARMIL单体最大化学计量比为两个CP杂二聚体，亲和力为~100 nM。考虑到这种亲和力，以及CARMIL(~2um)和CP(~1um)的细胞浓度，CARMIL将被预测为显著影响体内带刺末端的封顶。最近的体外实验表明，CARMIL：CP复合体可以覆盖肌动蛋白细丝，表明CARMIL不能简单地将CP隔离在不活跃的池中。相反，CARMIL和CP形成了一种新的带刺末端帽，它(I)可以被肌球蛋白I移位到带刺末端，并且(Ii)通过它也能够招募和激活Arp2/3，允许从带帽末端组装新的肌动蛋白细丝。

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.