Gascard P, Nunomura W, Lee G, Walensky L D, Krauss S W, Takakuwa Y, Chasis J A, Mohandas N, Conboy J G
Life Sciences Division, Department of Subcellular Structure, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Mol Biol Cell. 1999 Jun;10(6):1783-98. doi: 10.1091/mbc.10.6.1783.
The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.
红细胞膜细胞骨架蛋白4.1是一个遗传和拓扑结构复杂的家族的典型成员，该家族由组合性可变剪接途径产生，定位于包括细胞核在内的不同细胞内位点。为了探索核定位的分子决定因素，我们用表位标记的天然红细胞蛋白4.1（4.1R）异构体以及诱变和截短的衍生物转染COS-7细胞。4.1R80异构体高效核输入需要两个远距离的拓扑分选信号：一个由可变外显子16编码的碱性肽KKKRER，作为弱核心核定位信号（4.1R NLS），以及一个由可变外显子5编码的酸性肽EED。缺少这两个外显子之一的4.1R80异构体显示核输入减少。将各种4.1R80构建体与细胞质报告蛋白丙酮酸激酶融合，证实了完整NLS功能对这两个基序的需求。在重组Rch1（人输入蛋白α2）、输入蛋白β和GTP酶Ran存在的情况下，4.1R80在洋地黄皂苷通透的COS-7细胞核中被高效导入。使用共振镜检测技术对蛋白质-蛋白质相互作用进行定量分析表明，4.1R80在体外与Rch1以高亲和力结合（KD = 30 nM）。如果外显子5中的EED基序或外显子16中的4.1R NLS缺失或突变，亲和力分别至少降低7倍和20倍，证实这两个基序都是4.1R80高效输入蛋白介导的核输入所必需的。
