MAMMALIAN KARYOPHERIN ALPHA(1)BETA AND ALPHA(2)BETA HETERODIMERS - ALPHA(1) OR ALPHA(2) SUBUNIT BINDS NUCLEAR-LOCALIZATION SIGNAL AND BETA-SUBUNIT INTERACTS WITH PEPTIDE REPEAT-CONTAINING NUCLEOPORINS
MAMMALIAN KARYOPHERIN ALPHA(1)BETA AND ALPHA(2)BETA HETERODIMERS - ALPHA(1) OR ALPHA(2) SUBUNIT BINDS NUCLEAR-LOCALIZATION SIGNAL AND BETA-SUBUNIT INTERACTS WITH PEPTIDE REPEAT-CONTAINING NUCLEOPORINS
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DOI：
10.1073/pnas.92.14.6532
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发表时间：
1995-07-03
影响因子：
11.1
通讯作者：
RADU, A
中科院分区：
文献类型：
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作者：
MOROIANU, J;HIJIKATA, M;RADU, A
尽管与人核浆转运蛋白α（1）只有44%的相同性，但人核浆转运蛋白α（2）（Rch 1蛋白）在识别标准核定位序列和依赖于核浆转运蛋白β的靶向毛地黄皂苷透化细胞核膜方面取代了人核浆转运蛋白α（1）（hSRP-1/NPI-1）。通过甲醇固定细胞的免疫荧光显微镜，karyopherin β定位于细胞质和核膜，并且不存在于核内部。毛地黄皂苷透化布法罗大鼠肝细胞耗尽其内源性核转运蛋白β。重组核转运蛋白β可以在0 ℃下直接结合毛地黄皂苷透化细胞的核膜（对接反应）。相反，重组的核粒转运蛋白α（1）或α（2）不结合，除非存在核粒转运蛋白β。同样，在所有重组转运因子（核粒转运蛋白α（1）或α（2）、核粒转运蛋白β、Ran和p10）的输入反应（20 ℃）中，输入依赖于核粒转运蛋白β。在30分钟输入反应后，外源性添加的转运因子的定位显示，在核膜上有核浆转运蛋白β，在核内部有核浆转运蛋白α（1）或α（2）、Ran和p10。在用SDS/PAGE分辨的和硝化纤维素转移的核膜蛋白的重叠测定中，S-35标记的核转运蛋白β与至少四种含肽重复的核孔蛋白Nup 358、Nup 214、Nup 153和Nup 98结合。
Although only 44% identical to human karyopherin alpha(1), human karyopherin alpha(2) (Rch1 protein) substituted for human karyopherin alpha(1) (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degrees C (docking reaction). In contrast, recombinant karyopherin alpha(1) or alpha(2) did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha(1) or alpha(2), karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha(1) or alpha(2), Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, S-35-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins-Nup358, Nup214, Nup153, and Nup98.