We recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid–liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi–cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph’s negative charge, the results indicate that pi–cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi–cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.

中文翻译：

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突触素和突触素之间的多价静电π-阳离子相互作用负责凝聚

我们最近表明，突触素 (Syph) 和突触素 (Syn) 可以诱导液 - 液相分离 (LLPS) 聚集活细胞中的小突触样微泡，这很容易让人联想到 SV 簇。然而，由于它们之间没有物理相互作用，它们凝聚的潜在机制仍然未知。在这里，我们表明 Syph 和 Syn 之间的凝聚主要受 Syph C 末端 (Ct) 的酪氨酸残基和带正电的 Syn 之间的多价阳离子静电相互作用的控制。我们发现 Syph Ct 本质上是无序的，它单独可以通过在体外拥挤环境中以高浓度相互作用形成液滴，或者在通过用光敏 CRY2PHR 或活细胞中多聚体蛋白的亚基标记的额外相互作用的辅助下形成液滴. Syph Ct 包含 10 个重复序列，其中 9 个以酪氨酸开头，将 9 个酪氨酸突变为丝氨酸 (9YS) 完全消除了 Syph Ct 的相分离特性，表明酪氨酸介导的 pi 相互作用至关重要。我们进一步发现 9YS 突变未能与 Syn 凝聚，并且由于 9YS 保留了 Syph 的负电荷，结果表明，π-阳离子相互作用而不是简单的电荷相互作用是其凝聚的原因。