Evans L L, Lee A J, Bridgman P C, Mooseker M S
Department of Anatomy and Neurobiology, Washington University, St Louis, MO 63110, USA.
J Cell Sci. 1998 Jul 30;111 ( Pt 14):2055-66. doi: 10.1242/jcs.111.14.2055.
Myosin-V has been linked to actin-based organelle transport by a variety of genetic, biochemical and localization studies. However, it has yet to be determined whether myosin-V functions as an organelle motor. To further investigate this possibility, we conducted a biochemical and functional analysis of organelle-associated brain myosin-V. Using the initial fractionation steps of an established protocol for the purification of brain myosin-V, we isolated a population of brain microsomes that is approx. fivefold enriched for myosin-V, and is similarly enriched for synaptic vesicle proteins. As demonstrated by immunoelectron microscopy, myosin-V associates with 30-40% of the vesicles in this population. Although a majority of myosin-V-associated vesicles also label with the synaptic vesicle marker protein, SV2, less than half of the total SV2-positive vesicles label with myosin-V. The average size of myosin-V/SV2 double-labeled vesicles (90+/-45 nm) is larger than vesicles that label only with SV2 antibodies (60+/-30 nm). To determine if these vesicles are capable of actin-based transport, we used an in vitro actin filament motility assay in which vesicles were adsorbed to motility assay substrates. As isolated, the myosin-V-associated vesicle fraction was nonmotile. However, vesicles pre-treated with ice-cold 0.1% Triton X-100 supported actin filament motility at rates comparable to those on purified myosin-V. This dilute detergent treatment did not disrupt vesicle integrity. Furthermore, while this treatment removed over 80% of the total vesicle proteins, myosin-V remained tightly vesicle-associated. Finally, function-blocking antibodies against the myosin-V motor domain completely inhibited motility on these substrates. These studies provide direct evidence that vesicle-associated myosin-V is capable of actin transport, and suggest that the activity of myosin-V may be regulated by proteins or lipids on the vesicle surface.
通过各种遗传学、生物化学和定位研究，肌球蛋白-V已与基于肌动蛋白的细胞器运输联系起来。然而，肌球蛋白-V是否作为细胞器马达发挥作用尚未确定。为了进一步研究这种可能性，我们对与细胞器相关的脑肌球蛋白-V进行了生物化学和功能分析。利用已建立的脑肌球蛋白-V纯化方案的初始分级分离步骤，我们分离出了一群脑微粒体，其肌球蛋白-V富集了约五倍，突触小泡蛋白也同样富集。免疫电子显微镜显示，肌球蛋白-V与这群小泡中的30%-40%相关联。尽管大多数与肌球蛋白-V相关的小泡也用突触小泡标记蛋白SV2标记，但在总的SV2阳性小泡中，用肌球蛋白-V标记的不到一半。肌球蛋白-V/SV2双标记小泡的平均大小（90±45纳米）大于仅用SV2抗体标记的小泡（60±30纳米）。为了确定这些小泡是否能够进行基于肌动蛋白的运输，我们使用了体外肌动蛋白丝运动分析，其中小泡被吸附到运动分析底物上。刚分离出来时，与肌球蛋白-V相关的小泡部分是不运动的。然而，用冰冷的0.1% Triton X-100预处理的小泡支持肌动蛋白丝运动，其速率与纯化的肌球蛋白-V上的速率相当。这种稀释去污剂处理不会破坏小泡的完整性。此外，虽然这种处理去除了超过80%的总小泡蛋白，但肌球蛋白-V仍与小泡紧密相关。最后，针对肌球蛋白-V马达结构域的功能阻断抗体完全抑制了这些底物上的运动。这些研究提供了直接证据，表明与小泡相关的肌球蛋白-V能够进行肌动蛋白运输，并表明肌球蛋白-V的活性可能受小泡表面的蛋白质或脂质调节。
