Schaap P J, van't Riet J, Woldringh C L, Raué H A
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands.
J Mol Biol. 1991 Sep 5;221(1):225-37. doi: 10.1016/0022-2836(91)80216-h.
The regions of the large subunit ribosomal protein L25 from Saccharomyces cerevisiae responsible for nuclear localization of the protein were identified by constructing fusion genes encoding various segments of L25 linked to the amino terminus of beta-galactosidase. Indirect immunofluorescence of yeast cells expressing the fusions demonstrated that amino acid residues 1 to 17 as well as 18 to 41 of L25 promote import of the reporter protein into the nucleus. Both nuclear localization signal (NLS) sequences appear to consist of two distinct functional parts: one showed relatively weak nuclear targeting activity, whereas the other considerably enhances this activity but does not promote nuclear import by itself. Microinjection of in vitro prepared intact and N-terminally truncated L25 into Xenopus laevis oocytes demonstrated that the region containing the two NLS sequences is indeed required for efficient nuclear localization of the ribosomal protein. This conclusion was confirmed by complementation experiments using a yeast strain that conditionally expresses wild-type L25. The latter experiments also indicated that amino acid residues 1 to 41 of L25 are required for full functional activity of yeast 60 S ribosomal subunits. Yeast cells expressing forms of L25 that lack this region are viable, but show impaired growth and a highly abnormal cell morphology.
通过构建编码与β-半乳糖苷酶氨基末端相连的L25不同片段的融合基因，确定了酿酒酵母大亚基核糖体蛋白L25中负责该蛋白核定位的区域。对表达这些融合蛋白的酵母细胞进行间接免疫荧光检测表明，L25的第1至17位氨基酸残基以及第18至41位氨基酸残基可促进报告蛋白导入细胞核。这两个核定位信号（NLS）序列似乎都由两个不同的功能部分组成：一个显示出相对较弱的核靶向活性，而另一个则大大增强了这种活性，但自身并不促进核输入。将体外制备的完整L25和N端截短的L25显微注射到非洲爪蟾卵母细胞中表明，含有这两个NLS序列的区域确实是核糖体蛋白有效核定位所必需的。使用条件性表达野生型L25的酵母菌株进行的互补实验证实了这一结论。后一组实验还表明，L25的第1至41位氨基酸残基是酵母60 S核糖体亚基充分发挥功能活性所必需的。表达缺乏该区域的L25形式的酵母细胞是有活力的，但生长受损且细胞形态高度异常。
