Veit M, Becher A, Ahnert-Hilger G
Department of Immunology and Molecular Biology, Free University, Berlin, Germany.
Mol Cell Neurosci. 2000 Apr;15(4):408-16. doi: 10.1006/mcne.1999.0830.
Neuronal SNARE-proteins such as synaptobrevin, SNAP 25, and synaptotagmin are key players during neurosecretion. So far palmitoylation of SNAP-25 and synaptotagmin 1 have been described in vivo. Here we have analyzed palmitoylation of the SNARE-proteins synaptobrevin 2 and synaptotagmin in vitro using synaptosomal and synaptic vesicle preparations from rat brain. Labeling of synaptic vesicles prepared from adult brain with [3H]palmitate revealed synaptobrevin 2 besides synaptotagmin 1 as major palmitoylated proteins. [3H]Palmitoylation of synaptobrevin 2 was resistant to chloroform/methanol extraction, but sensitive to reducing agents indicating a covalent fatty acid bond to cysteine residues. Palmitoylation of synaptobrevin 2 was also confirmed using endogenous synaptobrevin 2 present in PC-12 cells and synaptobrevin 2 expressed with a vacciniavirus system in Cos cells. In contrast to the situation seen with membrane preparations obtained from adult brain, synaptic vesicles prepared from embryonic rat brain did not support [3H]palmitoylation of synaptobrevin and synaptotagmin. These results suggest, that both synaptobrevin 2 and synaptotagmin were efficiently palmitoylated from mature synaptic vesicles. However, at least one component of the palmitoylation machinery is developmentally upregulated.
神经元SNARE蛋白，如突触小泡蛋白、SNAP 25和突触结合蛋白，是神经分泌过程中的关键参与者。到目前为止，SNAP - 25和突触结合蛋白1的棕榈酰化已在体内得到描述。在这里，我们使用大鼠脑的突触体和突触小泡制剂，在体外分析了SNARE蛋白突触小泡蛋白2和突触结合蛋白的棕榈酰化。用[3H]棕榈酸标记成年大鼠脑制备的突触小泡，结果显示除了突触结合蛋白1外，突触小泡蛋白2也是主要的棕榈酰化蛋白。突触小泡蛋白2的[3H]棕榈酰化对氯仿/甲醇提取有抗性，但对还原剂敏感，这表明脂肪酸与半胱氨酸残基形成了共价键。使用PC - 12细胞中存在的内源性突触小泡蛋白2以及在Cos细胞中用痘苗病毒系统表达的突触小泡蛋白2，也证实了突触小泡蛋白2的棕榈酰化。与从成年大鼠脑获得的膜制剂情况不同，从胚胎大鼠脑制备的突触小泡不支持突触小泡蛋白和突触结合蛋白的[3H]棕榈酰化。这些结果表明，突触小泡蛋白2和突触结合蛋白在成熟的突触小泡中都能有效地进行棕榈酰化。然而，棕榈酰化机制的至少一个成分在发育过程中上调。
