Jans D A, Moll T, Nasmyth K, Jans P
Max-Planck-Institut für Biophysik, Frankfurt am Main, Federal Republic of Germany.
J Biol Chem. 1995 Jul 21;270(29):17064-7. doi: 10.1074/jbc.270.29.17064.
Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SWI5 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G1. beta-galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro- Arg-Lys655) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G1 or G2/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser microscopy. Cell cycle-dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser646 and Ser664) to alanine resulted in constitutive nuclear localization. In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS-dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold. We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.
对转录因子（TFs）核运输的控制代表了基因调控的一个水平，这对于细胞分化、转化和信号转导等细胞过程不可或缺。酿酒酵母转录因子SWI5以细胞周期依赖性方式被排除在细胞核外，这是由细胞周期蛋白依赖性激酶（cdk）CDC28磷酸化介导的。核进入发生在G1期。携带SWI5氨基酸633 - 682的β-半乳糖苷酶融合蛋白，包括核定位序列（NLS：Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro-Arg-Lys655），使用间接免疫荧光法在被阻滞在G1或G2/M期的合适温度敏感酵母菌株中分析其亚细胞定位，并通过向活的大鼠肝癌或非洲绿猴肾vero细胞中显微注射荧光标记的细菌表达蛋白和定量共聚焦激光显微镜分析其核输入动力学。酵母中细胞周期依赖性核定位既依赖于NLS也依赖于cdk位点，其中cdk位点丝氨酸（Ser646和Ser664）突变为丙氨酸会导致组成型核定位。在哺乳动物细胞中，SWI5融合蛋白同样以依赖NLS的方式转运到细胞核，而cdk位点丝氨酸突变为丙氨酸会使核积累的最大水平从约1倍增加到超过8倍。我们认为cdk位点的磷酸化抑制了SWI5的核运输，这与我们之前关于cdk cdc2磷酸化抑制SV40大T抗原核运输的观察结果一致。结果首次表明酵母NLS及其调控机制在高等真核生物中具有功能，这意味着蛋白质向细胞核运输的调控信号具有普遍性质。
