Interactions and three-dimensional localization of a group of nuclear pore complex proteins
Interactions and three-dimensional localization of a group of nuclear pore complex proteins
复制标题
一组核孔复合蛋白的相互作用和三维定位
作者：
Nelly Pant;Ricardo Bastos;Isabel McMorrow;Brian Burke
我们已经使用针对一些核孔复合物（NPC）蛋白的抗体来确定它们在NPC三维结构中的相互作用和位置。一种名为QE5的单克隆抗体在Western blots上识别了三种NPC多肽，p250， NUP153和p62，并通过免疫荧光显微镜标记了几种培养细胞系的核膜。这三个多肽含有o -连接的N-乙酰氨基葡萄糖残基，通过洗涤剂/高盐处理从NPC中释放出来，形成离散的高分子量配合物。p250被发现与一种新的75 kD蛋白相关，NUP153被释放为约1兆道尔顿的同型寡聚物，p62与58和54 kD的多肽相关（先前由Finlay， D. R.， E. Meier， P. Bradley， J. Horecka和D. J. Forbes报道）。[j] .细胞生物学杂志。P75、p58和p54在体外未被半乳糖化。用金共轭QE5标记爪蟾卵母细胞NEs，通过速冻/冷冻干燥/旋转金属阴影法制备电镜。这种EM制备方法使我们能够更精确地将抗体的表位定位到NPC的细胞质丝和核筐上。由于QE5可以识别3个o -链的NPC糖蛋白，我们将其标记与识别o -链n -乙酰氨基葡萄糖的凝集素小麦胚芽凝集素进行了比较。发现这两个探针产生相似的，虽然不完全相同，标签的分布。为了鉴定具有特定NPC成分的单个蛋白，我们使用了针对NUP153的抗肽抗体和单特异性抗p250多克隆抗体。用这两种抗体标记已证明NUP153是核筐的一个组成部分，其至少一个表位位于其末端环上，而p250是细胞质丝的一个组成部分。
We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N- acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold- conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.