Mutations within the Ran/TC4 GTPase - Effects on regulatory factor interactions and subcellular localization
Mutations within the Ran/TC4 GTPase - Effects on regulatory factor interactions and subcellular localization
复制标题
DOI：
10.1074/jbc.271.51.32834
复制
发表时间：
1996-12-20
影响因子：
4.8
通讯作者：
Macara, IG
中科院分区：
文献类型：
--
作者：
Lounsbury, KM;Richards, SA;Macara, IG
Ran是GTP酶Ras超家族的成员，主要定位于细胞核中，是蛋白质通过核孔主动转运的必要组分。Ran功能的破坏影响有丝分裂、DNA合成以及RNA加工和输出的调节。为了探索Ran功能的机制，对Ran GTPase的突变体进行了表征，其中一些突变体能够主要干扰核蛋白输入。与野生型Ran不同，推定的功能获得突变体（G19 V Ran）对交换因子RCC 1不敏感。此外，G19 V Ran和效应结构域突变体（L43 E和E46 G Ran）对GTP酶激活蛋白Fug 1不敏感。从转染的BHK 21细胞中分离的表位标记的G19 V Ran和L43 E Ran各自约50%与GTP结合，而野生型和C末端缺失突变体（Delta-DE Ran）主要与GDP结合。虽然G19 V Ran与已知的Ran结合蛋白和分离的Ran结合结构域相互作用，但T24 N Ran不相互作用，并且L43 E Ran的结合显著降低。在BHK 21细胞中表达的野生型HA 1标记的Ran是核的，而Ran的G19 V、T24 N、L43 E和E46 G形式主要定位于核膜，并且Delta-DE Ran主要是胞质的。当透化的BHK 21细胞与表达突变体的COS细胞的提取物一起孵育时，观察到类似的结果。因此，影响Ran与调节蛋白和效应物相互作用的突变可以破坏Ran的正常亚细胞定位，从而支持Ran介导的核输入的当前模型。
Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering with nuclear protein import, Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) was not sensitive to the exchange factor, RCC1. In addition the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activating protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells were each about 50% GTP-bound, whereas the wild-type and a C-terminal deletion mutant (Delta-DE Ran) were primarily bound to GDP. While G19V Ran interacted with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran did not, and binding by L43E Ran was substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells was nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran were predominantly localized at the nuclear envelope, and Delta-DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effecters can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.