Krementsova Elena B, Hodges Alex R, Lu Hailong, Trybus Kathleen M
Department of Molecular Physiology and Biophysics, University of Vermont, 149 Beaumont Avenue, Burlington, VT 05405, USA.
J Biol Chem. 2006 Mar 3;281(9):6079-86. doi: 10.1074/jbc.M510041200. Epub 2005 Dec 23.
Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.
非常规肌球蛋白V在从肌动蛋白丝上解离之前会沿其迈出许多36纳米的步长，从而确保其能够在细胞内长距离运输货物。在本研究中，我们通过分析小鼠肌球蛋白V与酵母非持续性V类肌球蛋白（Myo4p）的嵌合体的特性，评估了影响持续性运行长度的结构特征（雷克 - 彼得森，S. L.，泰斯卡，M. J.，诺维克，P. J.，和穆斯克，M. S.（2001年）《细胞生物学杂志》153卷，1121 - 1126页）。令人惊讶的是，在低盐条件下，一个在小鼠肌球蛋白V的颈部和杆部含有酵母运动结构域的嵌合体（Y - MD）显示出比小鼠野生型更长的运行长度。小鼠肌球蛋白V的运行长度对盐的依赖性很小，而Y - MD的运行长度则随离子强度急剧下降，类似于在小鼠肌球蛋白V主链中含有酵母环2的嵌合体。在循环的弱结合状态下，环2与肌动蛋白上的酸性斑块结合（福尔克曼，N.，刘，H.，黑兹尔伍德，L.，克雷门索娃，E. B.，洛伊，S.，特赖布斯，K. M.，和哈内因，D.（2005年）《分子细胞》19卷，595 - 605页）。与野生型的 +6相比，含有酵母环2的构建体在稳态ATP酶测定中对肌动蛋白显示出更高的K(m)值。结果表明带正电荷的环2和对肌动蛋白的高亲和力对于在生理离子强度附近维持持续性很重要。
