Matunis M J, Wu J, Blobel G
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA.
J Cell Biol. 1998 Feb 9;140(3):499-509. doi: 10.1083/jcb.140.3.499.
RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.
RanGAP1是Ran的GTP酶激活蛋白，Ran是一种小的类Ras GTP酶，参与调节核质运输。在脊椎动物中，RanGAP1以两种形式存在：一种是细胞质形式，另一种集中在核孔复合体（NPC）的细胞质纤维上。RanGAP1与NPC相关的形式通过小泛素样蛋白SUMO-1进行共价修饰，我们最近提出SUMO-1修饰的作用是将RanGAP1靶向到NPC。在这里，我们确定了RanGAP1中指定SUMO-1修饰的结构域，并证明该结构域中抑制修饰的突变也会抑制其靶向NPC。将异源蛋白靶向到NPC取决于指定SUMO-1修饰的决定因素，也取决于RanGAP1羧基末端结构域中的其他决定因素。发现SUMO-1修饰和这些其他决定因素指定了RanGAP1羧基末端结构域与核孔蛋白Nup358在Ran结合结构域三与四之间的区域之间的相互作用。这些发现共同表明，SUMO-1修饰通过在RanGAP1羧基末端结构域中暴露或创建一个Nup358结合位点，将RanGAP1靶向到NPC。令人惊讶的是，还发现RanGAP1的羧基末端结构域含有一个核定位信号。这个核定位信号以及九个富含亮氨酸的核输出信号基序的存在表明，RanGAP1可能在细胞核和细胞质之间穿梭。
