SNARE-catalyzed fusion events are regulated by Syntaxin1A-lipid interactions
SNARE-catalyzed fusion events are regulated by Syntaxin1A-lipid interactions
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DOI：
10.1091/mbc.e07-02-0148
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发表时间：
2008-02-01
影响因子：
3.3
通讯作者：
Stuenkel, Edward L.
中科院分区：
文献类型：
--
作者：
Lam, Alice D.;Tryoen-Toth, Petra;Stuenkel, Edward L.
膜融合是一个既涉及蛋白质又涉及脂质的过程。尽管最终克服融合能量障碍的SNARE蛋白已经被广泛研究，但对由特定膜脂决定的能量障碍本身的调节在很大程度上被忽视了。我们的发现揭示了SNARE蛋白的一种新功能，即通过直接结合和隔离融合部位的融合性脂类来降低融合的能量障碍。除了多种多聚磷脂外，我们还证明了Synaxin 1A与融合性脂质磷脂酸之间的特异性相互作用，并将Synaxin 1A内的一个多碱的膜旁区域定义为其脂结合区域。在PC-12细胞中，Synaxin1A突变导致脂质结合逐渐减少，导致诱发分泌逐渐减少。此外，对由脂质结合缺陷Synaxin1A突变体(5RK/A)驱动的融合事件的安培分析表明，融合孔动力学发生了变化，提示分泌过程中存在能量缺陷。磷脂酸产生酶磷脂酶D1的过表达完全挽救了5RK/A突变体的分泌缺陷。此外，抑制磷脂酶D1活性显著减少对照分泌，而5RK/A介导的分泌相对不受影响。综上所述，这些数据表明，Synaxin1A与脂质的相互作用是SNARE催化的融合事件能量学的关键决定因素。
Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipid-binding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid-binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics, suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events.