Moreland R B, Nam H G, Hereford L M, Fried H M
Proc Natl Acad Sci U S A. 1985 Oct;82(19):6561-5. doi: 10.1073/pnas.82.19.6561.
To identify a signal involved in transporting a ribosomal protein to the nucleus, we constructed hybrid genes encoding amino-terminal segments of yeast ribosomal protein L3 joined to the amino-terminal end of the entire Escherichia coli beta-galactosidase molecule. The subcellular locations of the corresponding hybrid proteins in yeast were determined by in situ immunofluorescence. The first 21 amino acids of L3 were sufficient to localize beta-galactosidase to the nucleus. This region shows limited homology to portions of other nuclear proteins identified as essential for their transport. Larger fusion proteins were also localized to the nucleus. However, a hybrid protein containing all but the 14 carboxyl-terminal amino acids from L3 initially failed to localize; this defect was corrected by inserting a glycine- and proline-containing bridge between the L3 and beta-galactosidase moieties. The renovated protein was able to associate with ribosomes, suggesting that, in addition to entering the nucleus, this hybrid polypeptide was assembled into 60S ribosomal subunits that were subsequently exported to the cytoplasm.
为了鉴定参与将核糖体蛋白转运至细胞核的信号，我们构建了杂种基因，这些基因编码与完整的大肠杆菌β-半乳糖苷酶分子的氨基末端相连的酵母核糖体蛋白L3的氨基末端片段。通过原位免疫荧光确定相应杂种蛋白在酵母中的亚细胞定位。L3的前21个氨基酸足以将β-半乳糖苷酶定位于细胞核。该区域与其他已确定对其转运至关重要的核蛋白部分显示出有限的同源性。更大的融合蛋白也定位于细胞核。然而，一种包含L3除14个羧基末端氨基酸外所有氨基酸的杂种蛋白最初未能定位；通过在L3和β-半乳糖苷酶部分之间插入一个含甘氨酸和脯氨酸的桥来纠正这一缺陷。修复后的蛋白能够与核糖体结合，这表明，除了进入细胞核外，这种杂种多肽还组装成60S核糖体亚基，随后被输出到细胞质中。
