Yoneda Y, Arioka T, Imamoto-Sonobe N, Sugawa H, Shimonishi Y, Uchida T
Exp Cell Res. 1987 Jun;170(2):439-52. doi: 10.1016/0014-4827(87)90319-3.
We studied the mechanism of transport of proteins into the nucleus using synthetic peptides containing the nuclear location signal sequence of Simian virus 40 (SV 40) large T-antigen. When chick erythrocytes containing a synthetic large T-antigen nuclear translocation signal were fused with SV 40-transformed human fibroblasts, the migration of native large T-antigen into the chick nuclei was suppressed. Migration of proteins detected by human specific antinuclear autoimmune antibody was not blocked. An analog of the nuclear location signal peptide did not inhibit entry of large T-antigen into the chick nuclei. This result suggests that the peptide blocked the migration of only native large T-antigen into the nucleus, and that the signal of the majority of nuclear proteins for nuclear transport is not the same as that of the large T-antigen. The synthetic peptide was conjugated chemically with bovine serum albumin (BSA) and introduced into the cytoplasm of cultured human cells by red blood cell ghost-mediated microinjection. The BSA-synthetic peptide conjugate was found predominantly in the nucleus within 2 h after its introduction into the cells. BSA conjugated with the cross-linking reagent alone was not transported into the nucleus. Acetylated synthetic peptide was not effective in promoting nuclear localization of BSA. Mild trypsin treatment of the BSA-synthetic peptide conjugate suppressed nuclear localization. Conjugates of the synthetic peptide with phycoerythrin (Mr about 150 kD) and with secretory IgA (Mr about 380 kD) were both found in the nucleus very shortly after their introduction into the cytoplasm. These results suggest that the synthetic peptide containing the nuclear location signal sequence provides exogenous proteins with the ability to migrate into the nucleus. But, since a conjugate of the synthetic peptide with IgM (Mr about 940 kD) did not migrate into the nucleus after its microinjection, there may be a size limit in nuclear transport of conjugated proteins.
我们使用含有猿猴病毒40（SV 40）大T抗原核定位信号序列的合成肽，研究了蛋白质进入细胞核的转运机制。当含有合成大T抗原核转位信号的鸡红细胞与SV 40转化的人成纤维细胞融合时，天然大T抗原向鸡细胞核的迁移受到抑制。人特异性抗核自身免疫抗体检测到的蛋白质迁移未被阻断。核定位信号肽的类似物不抑制大T抗原进入鸡细胞核。这一结果表明，该肽仅阻断天然大T抗原向细胞核的迁移，并且大多数核蛋白的核转运信号与大T抗原的不同。将合成肽与牛血清白蛋白（BSA）化学偶联，并通过红细胞血影介导的显微注射将其引入培养的人细胞的细胞质中。发现BSA - 合成肽偶联物在引入细胞后2小时内主要存在于细胞核中。仅与交联试剂偶联的BSA未转运到细胞核中。乙酰化的合成肽在促进BSA的核定位方面无效。对BSA - 合成肽偶联物进行温和的胰蛋白酶处理会抑制核定位。合成肽与藻红蛋白（分子量约150 kD）和分泌型IgA（分子量约380 kD）的偶联物在引入细胞质后很快都出现在细胞核中。这些结果表明，含有核定位信号序列的合成肽为外源蛋白质提供了迁移到细胞核的能力。但是，由于合成肽与IgM（分子量约940 kD）的偶联物在显微注射后未迁移到细胞核中，因此偶联蛋白的核转运可能存在大小限制。
