The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor.
The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor.
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DOI：
10.1042/bj20112044
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发表时间：
2012-04-01
期刊：
影响因子：
--
通讯作者：
Shin YK
中科院分区：
文献类型：
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作者：
Lai Y;Shin YK
Syt1（突触结合蛋白 1）是突触小泡融合的主要 Ca2+ 传感器。尽管已知 Syt1 可以与 SNARE（可溶性 N-乙基马来酰亚胺敏感融合蛋白-附着蛋白受体）复合物和膜结合，但 Syt1 调节囊泡融合的机制仍存在争议。在本研究中，我们使用体外脂质混合测定来研究蛋白脂质体融合中Ca2+依赖性Syt1功能。为了研究酸性脂质的作用，改变囊泡中带负电荷的 DOPS（1,2-二油酰-sn-甘油-3-磷酸-l-丝氨酸）的浓度。 Syt1 在没有 Ca2+ 的情况下刺激脂质混合 3-10 倍。然而，使用 Ca2+ 后，效果又增强了 2-5 倍。仅当 t-SNARE（目标 SNARE）侧存在过量 PS（磷脂酰丝氨酸）时，才会观察到这种 Ca2+ 依赖性刺激。如果 v-SNARE（囊泡 SNARE）侧存在相等或更多的 PS，则不会观察到 Ca2+ 依赖性刺激。我们发现浓度在 10 至 50 μM 之间的 Ca2+ 足以产生最大的增强作用。单囊泡融合实验表明Ca2+依赖性增强主要是在对接上，而对脂质混合的影响很小。因此，对于 Syt1 作为 Ca2+ 传感器的作用，电荷不对称性似乎很重要，这可能在引导 Syt1 有效反式结合质膜方面发挥作用。
Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+ -dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-l-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3–10-fold without Ca2+. However, with Ca2+ there was an additional 2–5-fold enhancement. This Ca2+ -dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+ -dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50 μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+ -dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.