Lounsbury K M, Richards S A, Carey K L, Macara I G
Department of Pathology and Comprehensive Cancer Center, University of Vermont, Burlington, Vermont 05405, USA.
J Biol Chem. 1996 Dec 20;271(51):32834-41. doi: 10.1074/jbc.271.51.32834.
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 effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.
Ran是GTP酶Ras超家族的成员之一，主要定位于细胞核，是蛋白质通过核孔进行主动运输的必要组成部分。Ran功能的破坏会影响有丝分裂、DNA合成以及RNA加工和输出的调控。为了探究Ran功能的机制，对Ran GTP酶的突变体进行了表征，其中一些能够显性干扰核蛋白的输入。与野生型Ran不同，推定的功能获得性突变体（G19V Ran）对交换因子RCC1不敏感。此外，G19V Ran和效应结构域突变体（L43E和E46G Ran）对GTP酶激活蛋白Fug1也不敏感。从转染的BHK21细胞中分离出的带有表位标签的G19V Ran和L43E 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G19V Ran与已知的Ran结合蛋白以及一个分离的Ran结合结构域相互作用，但T24N Ran则不然，并且L43E Ran的结合显著减少。在BHK21细胞中表达的野生型HA1标签的Ran定位于细胞核，而Ran的G19V、T24N、L43E和E46G形式主要定位于核膜，Delta-DE Ran主要位于胞质溶胶中。当用表达这些突变体的COS细胞提取物孵育通透化的BHK21细胞时，观察到了类似的结果。因此，影响Ran与调节蛋白和效应器相互作用的突变会破坏Ran正常的亚细胞定位，这为当前Ran介导的核输入模型提供了支持。
