Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
Center for Molecular Neurobiology (ZMNH), Department of Molecular Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Cytoskeleton (Hoboken). 2018 Sep;75(9):395-409. doi: 10.1002/cm.21474.
The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.
肌球蛋白 Va 基于肌动蛋白的运动将许多货物运输，包括小脑浦肯野神经元 (PNs) 的平滑内质网 (SER) 和黑素细胞中的黑素体。鉴定与这种肌球蛋白相互作用的蛋白质是理解其细胞功能的关键。为此，我们使用重组酶通过同源重组将一个串联亲和纯化 (TAP) 标签插入到小鼠 MYO5A 基因座中的起始密码子之后，该标签由蛋白 A 的免疫球蛋白 G 结合域、烟草蚀纹病毒切割位点和 FLAG 标签组成。重要的是，我们提供的证据表明，肌球蛋白 Va 的 TAP 标记版本（TAP-MyoVa）在 PN 中的 SER 运输和黑素细胞中的黑素体定位方面正常发挥作用。鉴于此和其他证据表明 TAP-MyoVa 具有完全的功能，我们直接从小鼠小脑纯化了它及其相关蛋白，并对样品进行了质谱分析。正如预期的那样，鉴定到了已知的肌球蛋白 Va 结合伴侣，如动力蛋白轻链。重要的是，还暂定鉴定到了许多新的相互作用蛋白，包括鸟嘌呤核苷酸结合蛋白 G(o)亚单位 alpha (Gnao1)，它是精神分裂症的生物标志物。一致地，针对 Gnao1 的抗体可免疫沉淀肌球蛋白 Va，并且 Gnao1 定位于 PN 树突棘取决于肌球蛋白 Va。这里创建的小鼠模型应有助于鉴定新的肌球蛋白 Va 结合伴侣，这反过来又应促进我们对这种重要肌球蛋白在体内所起作用的理解。
