Steel G J, Laude A J, Boojawan A, Harvey D J, Morgan A
The Physiological Laboratory, University of Liverpool, United Kingdom.
Biochemistry. 1999 Jun 15;38(24):7764-72. doi: 10.1021/bi990315v.
The SEC18 gene product is 48% identical to mammalian NSF (N-ethylmaleimide-sensitive fusion protein), and both proteins encode cytoplasmic ATPases which are essential for membrane traffic in yeast and mammalian cells, respectively. A wealth of biochemical analysis has led to the description of a model for the action of NSF; through its interaction with SNAPs (soluble NSF attachment proteins), NSF can associate with SNAP receptors (SNAREs) on intracellular membranes, forming 20S complexes. SNAPs then stimulate the intrinsic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is based almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membrane fusion is conserved in all eukaryotic cells. If so, the crucial biochemical properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here that Sec18p can specifically interact not only with Sec17p but also with its mammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-SNAP stimulation of NSF, although differences in temperature and N-ethylmaleimide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNAP to stimulate regulated exocytosis in mammalian cells. We conclude that the mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved from yeast to mammals.
SEC18基因产物与哺乳动物的NSF（N - 乙基马来酰亚胺敏感融合蛋白）有48%的同源性，这两种蛋白都编码细胞质ATP酶，分别对酵母和哺乳动物细胞中的膜运输至关重要。大量的生化分析得出了一个NSF作用模型的描述；通过与SNAPs（可溶性NSF附着蛋白）相互作用，NSF可与细胞内膜上的SNAP受体（SNAREs）结合，形成20S复合物。然后，SNAPs刺激NSF的内在ATP酶活性，导致20S复合物解体，这对随后的膜融合至关重要。尽管这个模型几乎完全基于对NSF和α - SNAP原始克隆的体外研究，但人们普遍认为这种膜融合机制在所有真核细胞中都是保守的。如果是这样，NSF和SNAPs的关键生化特性应该由它们的酵母同源物Sec18p和Sec17p共享。我们在此报告，使用纯化的重组蛋白，Sec18p不仅能与Sec17p特异性相互作用，还能与其哺乳动物同源物α - SNAP相互作用。这种相互作用导致Sec18p D1结构域ATP酶活性受到刺激，其动力学与α - SNAP刺激NSF的动力学相似，尽管在温度和N - 乙基马来酰亚胺敏感性方面观察到NSF和Sec18p之间存在差异。此外，Sec18p可与突触SNARE蛋白相互作用，并能与α - SNAP协同作用，刺激哺乳动物细胞中的调节性胞吐作用。我们得出结论，Sec18p和Sec17p具有NSF和SNAPs的机制特性，从而证明从酵母到哺乳动物，膜融合的生物化学过程是保守的。
