Sweet D J, Gerace L
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
J Cell Biol. 1996 Jun;133(5):971-83. doi: 10.1083/jcb.133.5.971.
Signal-dependent transport of proteins into the nucleus is a multi-step process mediated by nuclear pore complexes and cytosolic transport factors. One of the cytosolic factors, Ran, is the only GTPase that has a characterized role in the nuclear import pathway. We have used a mutant form of Ran with altered nucleotide binding specificity to investigate whether any other GTPases are involved in nuclear protein import. D125N Ran (XTP-Ran) binds specifically to xanthosine triphosphate (XTP) and has a greatly reduced affinity for GTP, so it is no longer sensitive to inhibition by nonhydrolyzable analogues of GTP such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). using in vitro transport assays, we have found that nuclear import supported by XTP-Ran is nevertheless inhibited by the addition of non-hydrolyzable GTP analogues. This in conjunction with the properties of the inhibitory effect indicates that at least one additional GTPase is involved in the import process. Initial characterization suggests that the inhibited GTPase plays a direct role in protein import and could be a component of the nuclear pore complex.
蛋白质信号依赖型入核转运是一个由核孔复合体和胞质转运因子介导的多步骤过程。胞质因子之一的Ran是唯一在核输入途径中具有明确作用的GTP酶。我们使用了一种核苷酸结合特异性改变的Ran突变体来研究是否有其他GTP酶参与核蛋白输入。D125N Ran（XTP-Ran）特异性结合三磷酸黄苷（XTP），对GTP的亲和力大大降低，因此它不再对GTP的不可水解类似物如鸟苷5'-O-（3-硫代三磷酸）（GTPγS）的抑制敏感。通过体外转运试验，我们发现添加不可水解的GTP类似物仍会抑制XTP-Ran支持的核输入。这与抑制作用的特性相结合表明，至少有一种额外的GTP酶参与了输入过程。初步表征表明，被抑制的GTP酶在蛋白质输入中起直接作用，可能是核孔复合体的一个组成部分。
