SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis
SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis
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DOI：
10.1073/pnas.091502398
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发表时间：
2001-05-08
影响因子：
11.1
通讯作者：
Gould, GW
中科院分区：
文献类型：
--
作者：
Chamberlain, LH;Burgoyne, RD;Gould, GW
脂筏是大多数细胞类型膜内存在的微域。这些富含胆固醇和鞘糖脂的膜微结构域与某些信号转导和膜交通途径的调节有关。为了研究脂筏在神经内分泌细胞中组织胞吐途径的可能性，我们检查了胞吐机制的蛋白质与从 PC12 细胞中纯化的脂筏的关联，目标可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (tSNARE) 蛋白突触蛋白 1A 和 25 kDa 的突触体相关蛋白 (SNAP25)都被发现在脂筏中高度富集(大约25倍)。囊泡 SNARE 囊泡相关膜蛋白 (VAMP)2 也存在于筏组分中，但这种恢复的程度是可变的。然而，进一步分析表明，大多数 VAMP2 与一类不同的筏相关，该筏具有与含有突触蛋白 1A 和 SNAP-25 的筏不同的去污剂溶解度特征。有趣的是，没有其他研究的分泌蛋白与脂筏显着相关，包括 SNARE 效应蛋白，例如 nSec1。化学交联实验表明，syntaxin1A/SNAP-25 异二聚体在筏组分和非筏组分中同等存在，而 Syntaxin1A/nSec1 复合物仅在非筏组分中检测到。 sos抗性测定表明，raft相关的syntaxin1A/SNAP-25异二聚体能够与VAMP2相互作用。最后，细胞胆固醇水平的降低降低了PC12细胞多巴胺胞吐调节的程度。所描述的结果表明 SNARE 蛋白与脂筏的相互作用对于胞吐作用很重要，并且可能允许分泌机制的结构和空间组织。
Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells, The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP25) were both found to be highly enriched in lipid rafts (approximate to 25-foId). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. sos-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2, Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.