Banaschewski C, Höhne-Zell B, Ovtscharoff W, Gratzl M
Anatomisches Institut der Technischen Universität München, Germany.
Biochemistry. 1998 Nov 24;37(47):16719-27. doi: 10.1021/bi981339p.
alpha-SNAP and NSF are thought to act as soluble factors, which transiently bind to a complex formed between syntaxin and SNAP-25 located at the plasma membrane and synaptobrevin at the secretory vesicle membrane, at the moment of exocytosis. Here we present data which permit the novel conclusion that alpha-SNAP and NSF are not soluble in adrenal chromaffin cells but are rather membrane-bound in particular to undocked chromaffin vesicles. Evidence for this new paradigm is derived from several experimental approaches. First, alpha-SNAP and NSF were found predominantly at cellular membranes and not in the cytosol of cracked chromaffin cells. Second, alpha-SNAP and NSF were not released from membranes by Mg2+ATP, which causes priming of vesicles. Third, immune electron microscopy and immunoblotting of chromaffin vesicles purified by immunoisolation or density gradient centrifugation revealed the presence of alpha-SNAP and NSF together with typical vesicular proteins such as synaptobrevin and synaptotagmin. In the sucrose gradient 30% alpha-SNAP and 27% NSF were recovered with chromaffin vesicles. Bound alpha-SNAP was quantified (14 molecules/vesicle), and binding was characterized with recombinant his6-tagged alpha-SNAP. Overlay blots revealed that alpha-SNAP is bound to vesicular SNAP-25 and endogenous NSF. Our data show that mature chromaffin vesicles already contain specifically bound alpha-SNAP and NSF before docking at the plasmalemma.
α-SNAP和NSF被认为作为可溶性因子，在胞吐作用发生时，它们短暂地结合到位于质膜的 syntaxin和SNAP-25以及分泌囊泡膜上的突触囊泡蛋白形成的复合物上。在此，我们展示的数据支持了一个新的结论：α-SNAP和NSF在肾上腺嗜铬细胞中并非可溶性的，而是特别地结合在未对接的嗜铬囊泡的膜上。这一新模式的证据来自几种实验方法。首先，α-SNAP和NSF主要存在于细胞膜上，而非破裂的嗜铬细胞的胞质溶胶中。其次，Mg2+ATP（可引发囊泡的启动）并不能使α-SNAP和NSF从膜上释放。第三，通过免疫分离或密度梯度离心纯化的嗜铬囊泡的免疫电子显微镜和免疫印迹显示，α-SNAP和NSF与典型的囊泡蛋白如突触囊泡蛋白和突触结合蛋白一同存在。在蔗糖梯度中，30%的α-SNAP和27%的NSF与嗜铬囊泡一起被回收。结合的α-SNAP被定量（14个分子/囊泡），并且用重组的his6标记的α-SNAP对其结合特性进行了表征。覆盖印迹显示α-SNAP与囊泡的SNAP-25和内源性NSF结合。我们的数据表明，成熟的嗜铬囊泡在对接至质膜之前就已经含有特异性结合的α-SNAP和NSF。
