Department of Neuroscience, Karolinska Institutet, S-171 77, Stockholm, Sweden.
J Neurosci. 2011 Sep 28;31(39):13972-80. doi: 10.1523/JNEUROSCI.1289-11.2011.
Eps15 homology domain-containing proteins (EHDs) are conserved ATPases implicated in membrane remodeling. Recently, EHD1 was found to be enriched at synaptic release sites, suggesting a possible involvement in the trafficking of synaptic vesicles. We have investigated the role of an EHD1/3 ortholog (l-EHD) in the lamprey giant reticulospinal synapse. l-EHD was detected by immunogold at endocytic structures adjacent to release sites. In antibody microinjection experiments, perturbation of l-EHD inhibited synaptic vesicle endocytosis and caused accumulation of clathrin-coated pits with atypical, elongated necks. The necks were covered with helix-like material containing dynamin. To test whether l-EHD directly interferes with dynamin function, we used fluid-supported bilayers as in vitro assay. We found that l-EHD strongly inhibited vesicle budding induced by dynamin in the constant presence of GTP. l-EHD also inhibited dynamin-induced membrane tubulation in the presence of GTPγS, a phenomenon linked with dynamin helix assembly. Our in vivo results demonstrate the involvement of l-EHD in clathrin/dynamin-dependent synaptic vesicle budding. Based on our in vitro observations, we suggest that l-EHD acts to limit the formation of long, unproductive dynamin helices, thereby promoting vesicle budding.
Eps15 同源结构域蛋白 (EHDs) 是保守的 ATP 酶，参与膜重塑。最近发现 EHD1 在突触释放位点富集，表明其可能参与突触囊泡的运输。我们研究了 Lamprey 巨大的网状脊髓突触中的 EHD1/3 同源物 (l-EHD) 的作用。l-EHD 通过免疫胶体金在靠近释放位点的内吞结构中被检测到。在抗体微注射实验中，l-EHD 的扰动抑制了突触囊泡的内吞作用，并导致网格蛋白包被的凹陷异常伸长，颈部长。这些颈部被含有动力蛋白的螺旋状物质覆盖。为了测试 l-EHD 是否直接干扰动力蛋白的功能，我们使用流体支持的双层膜作为体外测定。我们发现，在持续存在 GTP 的情况下，l-EHD 强烈抑制了由动力蛋白诱导的囊泡出芽。l-EHD 还抑制了 GTPγS 存在下由动力蛋白诱导的膜小管形成，这与动力蛋白螺旋组装有关。我们的体内结果表明 l-EHD 参与了网格蛋白/动力蛋白依赖性突触囊泡出芽。基于我们的体外观察，我们认为 l-EHD 作用是限制长的、无产物的动力蛋白螺旋的形成，从而促进囊泡出芽。
