Subramaniam V N, Krijnse-Locker J, Tang B L, Ericsson M, Yusoff A R, Griffiths G, Hong W
Membrane Biology Laboratory, National University of Singapore.
J Cell Sci. 1995 Jun;108 ( Pt 6):2405-14. doi: 10.1242/jcs.108.6.2405.
We have raised a monoclonal antibody (mAb) (HFD9) that detects a 28 kDa protein (p28) enriched in the Golgi membrane. p28 was localized to the perinuclear Golgi region in all cell lines thus far examined. Its Golgi localization was confirmed by its colocalization with Golgi markers using indirect immunofluorescence microscopy. Immunogold labelling demonstrates that the majority of p28 was localized on the cis-Golgi and its associated structures. Two independent experiments demonstrate that the p28 epitope recognized by mAb HFD9 is exposed to the cytosol. Extraction of Golgi membranes with a variety of reagents revealed that p28 behaves like an integral membrane protein. mAb HFD9 thus defines a novel 28 kDa integral membrane protein on the cis-Golgi. To our knowledge, p28 represents the first integral membrane protein of the Golgi system identified via the antibody approach whose epitope is cytoplasmically-oriented and highly-conserved. Monoclonal antibody HFD9 will thus provide a useful tool for further studies on the cis side of the Golgi, which is not well characterised due to the lack of good markers.
我们制备了一种单克隆抗体（mAb）（HFD9），它能检测到一种在高尔基体膜中富集的28 kDa蛋白质（p28）。到目前为止，在所有检测的细胞系中，p28都定位于核周高尔基体区域。通过间接免疫荧光显微镜观察，它与高尔基体标记物共定位，从而证实了其在高尔基体的定位。免疫金标记显示，大多数p28定位于顺面高尔基体及其相关结构上。两项独立实验表明，单克隆抗体HFD9识别的p28表位暴露于胞质溶胶中。用多种试剂对高尔基体膜进行抽提，结果显示p28表现得像一种整合膜蛋白。因此，单克隆抗体HFD9确定了一种新的位于顺面高尔基体上的28 kDa整合膜蛋白。据我们所知，p28是通过抗体方法鉴定出的高尔基体系统的首个整合膜蛋白，其表位面向细胞质且高度保守。因此，单克隆抗体HFD9将为进一步研究高尔基体顺面提供一个有用的工具，由于缺乏良好的标记物，高尔基体顺面目前尚未得到很好的表征。
