THE RAN/TC4 GTPASE-BINDING DOMAIN - IDENTIFICATION BY EXPRESSION CLONING AND CHARACTERIZATION OF A CONSERVED SEQUENCE MOTIF
THE RAN/TC4 GTPASE-BINDING DOMAIN - IDENTIFICATION BY EXPRESSION CLONING AND CHARACTERIZATION OF A CONSERVED SEQUENCE MOTIF
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DOI：
10.1073/pnas.92.8.3328
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发表时间：
1995-04-11
影响因子：
11.1
通讯作者：
MACARA, IG
中科院分区：
文献类型：
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作者：
BEDDOW, AL;RICHARDS, SA;MACARA, IG
Ran/TC 4是一种必需的核GT 4，参与DNA复制的起始、进入和离开有丝分裂以及通过核孔复合物的核RNA和蛋白质转运。这种功能的多样性表明Ran与大量下游靶标相互作用。使用覆盖试验，我们检测到一个家庭的推定的目标蛋白，与GTP结合的RAN。只有一个这样的蛋白质，HTF 9a/RanBP 1的序列是已知的。我们现在已经克隆了两个额外的RAN结合蛋白，允许识别一个独特的，高度保守的序列基序约150个残基。这个基序代表了一个最小的Ran结合域，它稳定了Ran的GTP结合状态。分离的结构域也作为Ran-GT β活化蛋白的共活化剂发挥作用。HTF 9a蛋白的Ran结合结构域内的保守残基的突变急剧降低Ran结合。RAN结合蛋白与表位标记的RAN细胞裂解物共免疫沉淀，表明这些蛋白质可能在体内关联。一个以前未知的秀丽隐杆线虫基因可以编码一个蛋白质（96 kDa）拥有两个RAN结合域。这种开放的阅读框架也包含与核孔蛋白的相似性，表明Ran与核孔复合物之间存在功能联系。
Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of down-stream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBP1, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of approximate to 150 residues. This motif represents a minimal Ran-binding domain that stabilizes the GTP-bound state of Ran. The isolated domain also functions as a coactivator of Ran-GTPase-activating protein. Mutation of a conserved residue within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimmunoprecipitated with epitope-tagged Ran from cell lysates, suggesting that these proteins may associate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. This open reading frame also contains similarities to nucleoporins, suggesting a functional link between Ran and nuclear pore complexes.