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（灰烬）的黑素细胞在肌球蛋白VA依赖性黑色素体捕获中被禁用，因为肌球蛋白与黑色素体表面的缔合取决于这种常驻黑色素体膜蛋白的存在。 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肌球蛋白VA的黑色素片胶囊同工型，而不是脑部切割的同工型。外显子D是其他黑色素特异性尾部外显子，不需要。同样，全长肌球蛋白VA与黑色素体共定位并在稀黑色素细胞中挽救黑色素体分布的能力绝对取决于外显子F的存在，因为黑色素细胞肌球蛋白VA和黑色素细胞肌球蛋白肌球蛋白va没有外显型肌动蛋白，而没有exon do Colocalize and Crosip and Colos ynot colocalize Myosy Myosy Myoste fa note Myoste fa note fa note fa reosal va note fa note fa note fa note fa note fa yosin va va或救援。这些结果表明，如果肌球蛋白VA和RAB27A是运动：受体对，则其物理缔合应具有外显子F依赖性。与此相一致的是，黑色素细胞洗涤剂中存在的Rab27a与覆盖的珠子结合，与纯化的，全长的，旗帜标记的黑色素细胞肌球蛋白VA和黑色素肌球蛋白VA缺乏外显子D，但不与黑素细胞肌球蛋白VA缺乏外素f brain f brain f brain myososin va。 GTP将与黑素细胞肌球蛋白VA结合的Rab27a的量减少了〜4倍。最后，肌球蛋白VA和RAB27A的稳定相互作用似乎至少需要一个额外的裂解物来衍生的因子，因为纯化的，加载GTP的RAB27A并未与肌球蛋白VA涂层的珠子明显结合。总之，这些结果牢固地确定，在黑色素体作为运动体的背景下，肌球蛋白VA和RAB27a的功能：受体对，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，并建议将肌球蛋白VA募集到黑色素体表面募集，应由控制Rab27a核苷酸状态的因素来调节黑色素体表面。 dictyostelium carmil蛋白充当连接封顶蛋白（CP）和ARP2/3复合物通过其SH3结构域的支架。为了进一步表征这种复合物，我们将acanthamoeba carmil纯化为同质性。分析性超速离心，电子显微镜和化学交联研究表明，卡米尔处于单体：二聚体平衡状态，缔合常数为〜1UM。关于卡米尔纯化的最引人注目的观察结果是，它与CP广泛相交。通过凝胶过滤对两者的完全解离需要温和的混沌剂或低pH（5.4）。纯化的CP对Carmil的重新介绍，每位Carmil Momomer的最大化学计量法，并具有约100 nm的亲和力。鉴于这种亲和力，并且Carmil（〜2 UM）和CP（〜1 UM）的细胞浓度将被预测会显着影响体内刺的末端封盖。最近的体外实验表明，Carmil：CP复合物可以限制肌动蛋白丝，表明Carmil不能简单地将CP隔离在非活动库中。相反，Carmil和CP形成了一个新颖的带状端盖，该端盖（i）可以通过肌球蛋白I将其转移到带刺的末端，并且（ii）可以通过它也能够募集和激活ARP2/3的能力，允许将新的Actin丝组装从封口的末端组装。