McPherson P S, Takei K, Schmid S L, De Camilli P
Department of Cell Biology, Howard Hughes Medical Research Institute, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1994 Dec 2;269(48):30132-9.
Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A. J., Chilcote, T. J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6486-6490). We have now used antibodies raised against p145 which had been purified by Grb2 affinity chromatography and SDS-polyacrylamide gel electrophoresis to characterize this protein. p145 is neuron-specific and co-distributes with dynamin in subcellular fractions of rat brain. Both proteins are partially recovered in soluble and particulate fractions. By immunofluorescence, p145 is closely co-localized with dynamin, which we show here to be highly concentrated in nerve terminals. In contrast to synapsin I, which is highly enriched in a purified synaptic vesicle fraction, both p145 and dynamin are de-enriched in this fraction. However, both proteins are found on membranes which are immunoisolated with antibodies directed against the synaptic vesicle membrane protein synaptophysin, suggesting that dynamin and p145 are localized in part on organelles which represent intermediate stages in the reformation of synapatic vesicles during recycling. Finally, we have determined that p145, like dynamin, is a phosphoprotein which undergoes dephosphorylation in response to nerve terminal depolarization. These findings suggest that p145 participates with dynamin in synaptic vesicle endocytosis and recycling.
最近发现三种主要的大鼠脑蛋白能与Grb2的SH3结构域结合：突触蛋白I、发动蛋白和一种新的145 kDa蛋白（p145）（麦克弗森，P.S.，切尔尼克，A.J.，奇尔科特，T.J.，奥诺弗里，F.，本费纳蒂，F.，格林加德，P.，施莱辛格，J.，和德卡米利，P.（1994年）《美国国家科学院院刊》91，6486 - 6490）。我们现在使用针对通过Grb2亲和层析和SDS - 聚丙烯酰胺凝胶电泳纯化的p145产生的抗体来表征这种蛋白。p145是神经元特异性的，并且与发动蛋白在大鼠脑的亚细胞组分中共分布。这两种蛋白在可溶性和颗粒性组分中都有部分回收。通过免疫荧光，p145与发动蛋白紧密共定位，我们在此表明发动蛋白高度集中在神经末梢。与在纯化的突触小泡组分中高度富集的突触蛋白I相反，p145和发动蛋白在该组分中都减少了富集。然而，这两种蛋白都存在于用针对突触小泡膜蛋白突触素的抗体进行免疫分离的膜上，这表明发动蛋白和p145部分定位于在回收过程中代表突触小泡重塑中间阶段的细胞器上。最后，我们确定p145与发动蛋白一样，是一种磷蛋白，其会响应神经末梢去极化而发生去磷酸化。这些发现表明p145与发动蛋白一起参与突触小泡的内吞作用和回收。
