YEAST NUCLEAR-ENVELOPE PROTEINS CROSS REACT WITH AN ANTIBODY AGAINST MAMMALIAN PORE COMPLEX PROTEINS
YEAST NUCLEAR-ENVELOPE PROTEINS CROSS REACT WITH AN ANTIBODY AGAINST MAMMALIAN PORE COMPLEX PROTEINS
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DOI：
10.1083/jcb.108.6.2059
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发表时间：
1989-06-01
影响因子：
7.8
通讯作者：
BLOBEL, G
中科院分区：
文献类型：
--
作者：
ARIS, JP;BLOBEL, G
我们利用一种针对大鼠肝核蛋白的单克隆抗体对酵母核中的两种交叉反应蛋白进行了研究。在大鼠肝脏中，这种单克隆抗体mAb 414与核孔复合物蛋白结合，其中一个分子量为62,000 （Davis， L. I. and G. Blobel. 1987）。Proc。国家的。学会科学。美国。84:7552 - 7556)。在酵母中，mAb 414通过免疫印迹与两种蛋白交叉反应，这两种蛋白的表观分子量分别为110,000和95,000，分别称为p110和p95。免疫印迹法检测亚细胞部分显示p110和p95只位于细胞核部分。mAb 414从酵母细胞粗提取物中提取多种蛋白质，包括p110、p95和a .apprx。55-kD蛋白质。亚细胞部分的免疫沉淀纯化核只产生p110和p95，而.apprx。从可溶性部分免疫沉淀55-kD蛋白。纯化后的细胞核用dna酶消化生成核包膜，释放出部分p110，但大部分p110只有在包膜用1 M NaCl处理后才被溶解。利用酵母细胞和分离细胞核的免疫荧光定位显示细胞核的点状和斑片状染色模式。共聚焦激光扫描免疫荧光显微镜较好地解决了点状和斑状染色模式，并显示了核膜上的荧光区域。利用纯化细胞核和mAb 414进行的包埋后免疫金电镜显示，酵母核膜上有胶体金修饰，但对孔复合物的解析能力较差，无法实现进一步的超微结构定位。细胞核免疫金标记后包埋提示孔复合物的修饰。因此，p110和/或p95定位于酵母的核膜，并且可能是核孔复合物的组成部分。
We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a .apprx. 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the .apprx. 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilzed only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.