Gu Z, Moerschell R P, Sherman F, Goldfarb D S
Department of Biology, University of Rochester, NY 14627.
Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10355-9. doi: 10.1073/pnas.89.21.10355.
Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.
在细胞色素c的N端添加了核定位信号后,它被错误定位于细胞核,导致酵母菌株中线粒体细胞色素c缺乏。该菌株的回复突变使得能够分离出在核转运方面有缺陷的温度条件突变体,其中一个突变体nip1-1就证明了这一点,它在含有酵母核糖体蛋白L29核定位信号的LacZ蛋白的核积累方面存在缺陷。NIP1⁺基因被克隆出来,结果显示它编码一种93,143道尔顿的蛋白质。此外,一种带有表位标签的NIP1蛋白在SDS/聚丙烯酰胺凝胶中迁移时的质量约为100,000道尔顿,免疫荧光显示它主要定位于细胞质中。通过基因破坏实验表明NIP1⁺是一个必需基因。有趣的是,NIP1有一个富含丝氨酸的酸性N端区域,在这方面与先前描述的一种核定位信号结合蛋白NSR1的N端区域相似。