A nuclear localization signal binding protein in the nucleolus.
A nuclear localization signal binding protein in the nucleolus.
复制标题
DOI：
10.1083/jcb.111.6.2235
复制
发表时间：
1990-12
期刊：
影响因子：
--
通讯作者：
Blobel G
中科院分区：
文献类型：
--
作者：
Meier UT;Blobel G
我们使用与人血清白蛋白（肽缀合物）交联的 SV-40 T 抗原的功能性野生型和突变型合成核定位信号肽，在蛋白质印迹上测定其与大鼠肝细胞核蛋白质的结合。 140 和 55 kD（p140 和 p55）的蛋白质仅被野生型肽缀合物识别。游离野生型肽竞争与p140和p55结合的野生型肽缀合物，而与野生型只有一个氨基酸不同的游离突变肽，竞争效率较低。这两种蛋白质可以通过低或高离子强度缓冲液从细胞核中提取。我们纯化了 p140，并在鸡体内产生了针对从聚丙烯酰胺凝胶上切下的蛋白质的多克隆抗体。根据抗 p140 抗体与全细胞亚细胞部分的蛋白质印迹上 140 kD 的单个核蛋白条带的反应性来判断，抗 p140 抗体是单特异性的。使用抗 p140 抗体对固定和透化的水牛肝 (BRL) 细胞进行间接免疫荧光显微镜检查，显示出明显的点状核仁染色。在固定和透化的 BRL 细胞上，罗丹明标记的野生型肽缀合物也以类似的模式与核仁结合。根据生化特征，p140 是一种新型核仁蛋白。 p140可能在核仁和细胞质之间穿梭，并充当核输入载体。
We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.