Ikonen E, de Almeid J B, Fath K R, Burgess D R, Ashman K, Simons K, Stow J L
European Molecular Biology Laboratory, D-69012, Heidelberg, Germany.
J Cell Sci. 1997 Sep;110 ( Pt 18):2155-64. doi: 10.1242/jcs.110.18.2155.
A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.
在真核细胞中，多种外周膜蛋白在运输小泡的出芽和运输过程中与高尔基体膜动态结合。一种针对高尔基体膜产生的单克隆抗体（AD7）识别一种外周膜蛋白p200，它与从反式高尔基体网络（TGN）出芽的小泡相关联。基于初步发现，研究了p200与高尔基体膜上肌球蛋白之间的潜在关联。使用针对鸡刷状缘非肌肉肌球蛋白II产生的抗体，在多种固定条件下对培养细胞进行免疫荧光染色。我们发现，除了存在于细胞质中或与应力纤维相关外，非肌肉肌球蛋白II还特异性地定位于高尔基体膜上。通过免疫印迹和电子显微镜水平的免疫金标记也在高尔基体膜上检测到了肌球蛋白II，发现它集中在源自高尔基体的小泡上。肌球蛋白II与高尔基体膜的关联是动态的，并且发现在G蛋白激活后增强。布雷菲德菌素A（BFA）也破坏了高尔基体膜上的肌球蛋白II染色。AD7和肌球蛋白II抗体在光学和电子显微镜水平的共定位促使我们研究两种抗体识别的200 kDa蛋白的性质。从MDCK细胞中分离出由AD7抗体免疫沉淀的200 kDa蛋白并用于微量测序。氨基酸序列数据使我们能够将p200鉴定为非肌肉肌球蛋白IIA的重链。此外，对AD7抗体在HeLa细胞提取物中特异性识别的另一种额外蛋白（240 kDa）进行了测序，并鉴定为另一种肌动蛋白结合蛋白细丝蛋白。这些结果表明，非肌肉肌球蛋白II与高尔基体膜相关联，并且与小泡相关的蛋白p200本身就是肌球蛋白II的重链。
