Saitoh H, Cooke C A, Burgess W H, Earnshaw W C, Dasso M
Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-5430, USA.
Mol Biol Cell. 1996 Sep;7(9):1319-34. doi: 10.1091/mbc.7.9.1319.
Ran is a small GTPase that is required for protein import, mRNA export, and the maintenance of nuclear structures. To gain a better understanding of Ran's role in the nucleus, we have sought to use Xenopus egg extracts for the purification and characterization of proteins from egg extracts bound with a high affinity to a glutathione-S-transferase-Ran fusion protein (GST-Ran). We found that GST-Ran associates specifically with at least 10 extract proteins. We determined the identifies of six Ran-interacting proteins (Rips), and found that they include RanBP2/Nup358, Nup153, Importin beta, hsc70, RCC1, and RanBP1. On the basis of peptide sequence, a seventh Rip (p88) seems to be similar but not identical to Fug1/RanGAP1, the mammalian Ran-GTPase-activating protein. Gel filtration analysis of endogenous extract proteins suggests that Importin beta acts as a primary GTP-Ran effector. Both Ran and Importin beta are coimmunoprecipitated by anti-p340RanBP2 antibodies in the presence of nonhydrolyzable GTP analogues, suggesting that Ran-Importin beta complexes interact with p340RanBP2. Two other Rips, p18 and p88, are coprecipitated with p340RanBP2 in a nucleotide-independent manner. Analysis of the Ran-GTPase pathway in Xenopus extracts allows the examination of interactions between Ran-associated proteins under conditions that resemble in vivo conditions more closely than in assays with purified components, and it thereby allows additional insights into the molecular mechanism of nuclear transport.
Ran是一种小GTP酶，在蛋白质导入、mRNA输出以及核结构维持过程中发挥作用。为了更深入了解Ran在细胞核中的作用，我们尝试利用非洲爪蟾卵提取物来纯化并鉴定与谷胱甘肽-S-转移酶-Ran融合蛋白（GST-Ran）具有高亲和力的卵提取物中的蛋白质。我们发现GST-Ran特异性地与至少10种提取物蛋白相结合。我们确定了6种Ran相互作用蛋白（Rips）的身份，发现它们包括RanBP2/Nup358、Nup153、输入蛋白β、hsc70、RCC1和RanBP1。根据肽序列，第七种Rip（p88）似乎与Fug1/RanGAP1（哺乳动物Ran-GTP酶激活蛋白）相似但并不相同。对内源提取物蛋白的凝胶过滤分析表明，输入蛋白β作为主要的GTP-Ran效应器发挥作用。在不可水解的GTP类似物存在的情况下，Ran和输入蛋白β均被抗p340RanBP2抗体共免疫沉淀，这表明Ran-输入蛋白β复合物与p340RanBP2相互作用。另外两种Rips，p18和p88，以不依赖核苷酸的方式与p340RanBP2共沉淀。对非洲爪蟾提取物中Ran-GTP酶途径的分析能够在比使用纯化成分的实验更接近体内条件的情况下，研究Ran相关蛋白之间的相互作用，从而能够更深入地了解核运输的分子机制。
