To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However, versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable, as the fast dynamics of synaptic vesicle cycling difficult specific isolation trafficking proteins during such a transient surface exposure. Here we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity, enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept, we further applied this technology to obtain direct evidence of the surface translocation of non-canonical trafficking proteins, such as ATG9A and NPTX1, which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity, helping to define the molecular machinery that sustains neurotransmission in the mammalian brain.

中文翻译：

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通过邻近标记进行活动驱动的内源突触蛋白运输

为了在大脑中传输信息，突触小泡与突触前膜融合，释放其内容物并短暂暴露大量的小泡跨膜蛋白。然而，用于量化神经元活动期间内源蛋白质突触易位的通用方法仍然不可用，因为突触小泡循环的快速动态在这种短暂的表面暴露期间难以特异性分离运输蛋白质。在这里，我们开发了一种使用突触间隙邻近标记来捕获和量化突触处活动驱动的内源突触蛋白运输的新方法。我们表明，加速裂口生物素化时间以匹配囊泡胞吐作用的快速动力学，可以捕获神经活动期间短暂暴露在突触表面的内源性蛋白质，从而首次能够研究几乎所有内源性突触蛋白质的易位。作为概念验证，我们进一步应用该技术来获得非规范运输蛋白（例如 ATG9A 和 NPTX1）表面易位的直接证据，这些蛋白质已被提议在活动期间进行运输，但尚未得到直接证据。显示。这里介绍的技术进步将有助于未来的研究，剖析活动过程中突触胞吐蛋白质的分子特性，有助于定义维持哺乳动物大脑神经传递的分子机制。