Morgan J R, Prasad K, Hao W, Augustine G J, Lafer E M
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Neurosci. 2000 Dec 1;20(23):8667-76. doi: 10.1523/JNEUROSCI.20-23-08667.2000.
Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.
尽管衔接蛋白（APs）介导的网格蛋白组装在突触小泡循环中起主要作用，但APs介导网格蛋白组装的分子机制仍不清楚。我们在此证明，AP180与AP-2和AP-3一样，能与网格蛋白的N端结构域结合。序列分析揭示了一个包含DLL序列的基序，该基序在许多网格蛋白APs中以多个拷贝形式存在。逐步删除这些基序会导致AP180在体外组装网格蛋白的能力逐渐降低。含有该基序的AP180或AP-2的肽也竞争性抑制这两种蛋白介导的网格蛋白组装。将这些肽显微注射到鱿鱼巨大突触前终末中，可通过阻止质膜上包被小窝的形成，可逆性地阻断突触传递并抑制突触小泡内吞作用。这些结果表明，DLL基序赋予AP180和AP-2以及可能其他APs组装网格蛋白的特性。我们提出，APs通过APs中重复的DLL基序与网格蛋白N端结构域上的互补结合位点之间的多价相互作用，交联网格蛋白三脚蛋白复合体，从而促进网格蛋白组装。这些结果揭示了网格蛋白组装的结构基础，并为网格蛋白介导的突触小泡内吞作用的分子机制提供了新见解。
