Görlich D, Kostka S, Kraft R, Dingwall C, Laskey R A, Hartmann E, Prehn S
Wellcome/CRC Institute, Cambridge, UK.
Curr Biol. 1995 Apr 1;5(4):383-92. doi: 10.1016/s0960-9822(95)00079-0.
Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide.
We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors.
Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.
选择性蛋白质导入细胞核过程分两步进行：首先与核膜结合，随后通过核孔复合体进行能量依赖的转运。一种60kD的蛋白质——输入蛋白，对第一步核输入至关重要，而小G蛋白Ran/TC4对第二步至关重要。我们之前已将60kD的输入蛋白（输入蛋白60）纯化得到单一多肽。
我们鉴定出输入蛋白90，一种90kD的第二亚基，在镍（II）-次氮基三乙酸-琼脂糖亲和层析过程中，它会与输入蛋白60解离，该技术最初用于纯化输入蛋白60。对非洲爪蟾输入蛋白90进行部分氨基酸测序后，我们得以克隆并测序其人类同源物；输入蛋白90的氨基酸序列在这两个物种间具有显著的保守性。我们还从数据库条目中鉴定出一个同源的芽殖酵母序列。输入蛋白90增强了输入蛋白60对核蛋白输入的作用，表明输入蛋白复合体是负责输入的生理单位。为评估核定位序列是否被胞质受体蛋白识别，将与牛血清白蛋白相连的生物素标记的核定位信号偶联物与非洲爪蟾卵提取物中的胞质蛋白形成复合物；然后用链霉亲和素-琼脂糖回收复合物。结合蛋白的模式出人意料地简单，仅显示两条主要条带：输入蛋白复合体的条带。我们还表达了输入蛋白60的人类同源物Rch1p，并发现它在功能测定中能够替代其非洲爪蟾对应物。我们讨论了输入蛋白60和输入蛋白90与其他核输入因子的关系。
输入蛋白由一个60kD和一个90kD的亚基组成。它们共同构成核定位信号的胞质受体，使输入底物能够与核膜结合。
