Reassessment of the subcellular localization of p63.
Reassessment of the subcellular localization of p63.
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DOI：
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发表时间：
1995-06
影响因子：
4
通讯作者：
A. Schweizer;J. Rohrer;J. Slot;H. Geuze;S. Kornfeld
中科院分区：
文献类型：
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作者：
A. Schweizer;J. Rohrer;J. Slot;H. Geuze;S. Kornfeld
p63是一种II型整体膜蛋白，以前曾被认为是内质网和高尔基体之间膜网络的驻留蛋白。在本研究中，我们制备了一种针对纯化的人p63蛋白的多克隆抗体，通过共聚焦免疫荧光、免疫电镜和细胞分离来重新评估p63的亚细胞分布。COS细胞的双免疫荧光显示p63和含有kdel的管腔内质网标记蛋白明显共定位，但外核膜染色存在差异。转染p63的天然HepG2细胞和COS细胞的免疫电镜显示，内源性和过表达的p63主要定位于粗质内质网。虽然p63与内质网标记蛋白二硫异构酶共定位，但p63与内质网-高尔基中间区已确定的标记ERGIC-53几乎没有重叠。当从大鼠肝脏制备粗糙和光滑的膜时，发现p63与粗糙的内质网蛋白核糖素II共化。p63和核糖素II主要在粗糙的微粒体中被回收，并且大部分与中间的室标记蛋白p58分离。由此得出p63定位于粗糙内质网的结论。
p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.