RAPID PLASMENYLETHANOLAMINE-SELECTIVE FUSION OF MEMBRANE BILAYERS CATALYZED BY AN ISOFORM OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE - DISCRIMINATION BETWEEN GLYCOLYTIC AND FUSOGENIC ROLES OF INDIVIDUAL ISOFORMS
RAPID PLASMENYLETHANOLAMINE-SELECTIVE FUSION OF MEMBRANE BILAYERS CATALYZED BY AN ISOFORM OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE - DISCRIMINATION BETWEEN GLYCOLYTIC AND FUSOGENIC ROLES OF INDIVIDUAL ISOFORMS
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DOI：
10.1021/bi00038a013
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发表时间：
1995-09-26
期刊：
影响因子：
2.9
通讯作者：
GROSS, RW
中科院分区：
文献类型：
--
作者：
GLASER, PE;GROSS, RW
最近，我们证明了纤浆乙醇胺结构中固有的独特立体电子关系促进了膜融合，并且我们假设存在一种膜融合蛋白，它可以利用纤浆乙醇胺分子种类的倾向来适应倒六角相[Glaser & Gross (1994) Biochemistry 33， 5805-5812]。我们现在报道了兔脑细胞质中的隐性膜融合活性，需要与内源性抑制剂分离才能表达其活性，并证明该蛋白催化的囊泡融合对含有纤溶酶乙醇胺的膜囊泡具有高度选择性。通过连续柱色谱将胞浆蛋白催化膜融合活性纯化至表观均一性，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和银染色后显示单个38-kDa蛋白条带。自动 Edman 降解证明纯化的蛋白质是 3-磷酸甘油醛脱氢酶 (GAPDH) 的同工型，这一点通过使用多克隆抗体的蛋白质印迹分析以及针对 GAPDH 的单克隆抗体对膜融合活性的溶液状态灭活得到了证实。 GTP 亲和色谱和 Mono Q 色谱均将催化脱氢酶活性的 GAPDH 同工型与催化膜融合活性的 GAPDH 同工型分离。纯化的融合蛋白不依赖于钙，对 N-乙基马来酰亚胺处理具有抗性，并且对纤浆乙醇胺和胆固醇具有强制性要求。对纤浆乙醇胺促进的膜融合进行高分辨率停流动力学分析表明，GAPDH 亚型的一个四聚体每毫秒（平均）催化两个含有纤浆乙醇胺的囊泡之间发生一次融合事件。总的来说，这些结果构成了对一种蛋白质的首次描述，该蛋白质可以以满足观察到的体内突触小泡与突触前膜融合速率所施加的数学限制的速率催化小泡融合。
Recently we demonstrated that the unique stereoelectronic relationships inherent in the structure of plasmenylethanolamine facilitate membrane fusion, and we postulated the existence of a membrane fusion protein which could exploit the propensity of plasmenylethanolamine molecular species to adapt an inverted hexagonal phase [Glaser & Gross (1994) Biochemistry 33, 5805-5812]. We now report a cryptic membrane fusion activity in rabbit brain cytosol, which requires separation from an endogenous inhibitor to express its activity, and demonstrate that vesicle fusion catalyzed by this protein is highly selective for membrane vesicles containing plasmenylethanolamine. The cytosolic protein catalyzing membrane fusion activity was purified to apparent homogeneity by sequential column chromatographies, revealing a single 38-kDa protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Automated Edman degradation demonstrated that the purified protein is an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was confirmed by Western blot analysis utilizing polyclonal antibodies and by solution-state inactivation of membrane fusion activity by a monoclonal antibody directed against GAPDH. Both GTP-affinity and Mono Q chromatographies resolved GAPDH isoforms that catalyzed dehydrogenase activity from the GAPDH isoform that catalyzed membrane fusion activity. The purified fusion protein was calcium-independent, resistant to treatment with N-ethylmaleimide, and possessed an obligatory requirement for plasmenylethanolamine and cholesterol. High-resolution stopped-flow kinetic analysis of plasmenylethanolamine-facilitated membrane fusion demonstrated that one tetramer of the GAPDH isoform catalyzed one fusion event between two vesicles containing plasmenylethanolamine every millisecond (on average). Collectively, these results constitute the first description of a protein which can catalyze the fusion of vesicles at a rate which satisfies the mathematical constraints imposed by the observed rates of fusion of synaptic vesicles with the presynaptic membrane in vivo.