The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited
The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited
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DOI：
10.1038/sj.onc.1200949
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发表时间：
1997-03-27
期刊：
影响因子：
8
通讯作者：
Peters, R
中科院分区：
文献类型：
--
作者：
Middeler, G;Zerf, K;Peters, R
人p53基因在大肠杆菌中表达。大肠杆菌，纯化，标记与荧光素碘乙酰胺（IAF）和其特征在于序列特异性DNA结合和表位处置。将IAF-p53注射到3 T3细胞的胞质或胞核中，在数分钟内IAF-p53被输入或输出细胞核。共注射凝集素麦胚凝集素（WGA）抑制输入。相反，携带SV 40 T抗原的核定位序列（NLS）的肽-蛋白缀合物NLS-HSA仅输入而不输出。将IAF-p53注入3 T3多核体，并通过扫描显微光解进行光漂白，使得每个多核体仅单个核保持未漂白。发现IAF-p53从未漂白的核迅速迁移（半衰期类似于10分钟）到漂白的核，而NLS-HSA没有。毛地黄皂苷透化的细胞中，IAF-p53被导入细胞核。当核积累后从培养基中取出时，IAF-p53从核中输出。IAF-p53的核输入和输出都是快速的（几分钟的半衰期，22 ℃），并且被WGA或冰上孵育强烈抑制。NLS-HSA只进口，不出口。我们的结论是，核质运输的p53，相反，NLS-HSA，是双向的，在两个方向的运输是载体介导的和能量依赖。这些结果表明，p53含有核输出信号（内斯），除了输入信号（NLS），从而打开了新的观点的p53细胞组分的潜在调控。
Human p53 was expressed in E. coli, purified, labeled with fluorescein iodoacetamide (IAF) and characterized for sequence-specific DNA binding and epitope disposition. Injected into the cytoplasm or nuclei of 3T3 cells IAF-p53 was imported into or exported from nuclei within minutes. Import was inhibited by coinjection of the lectin wheat germ agglutinine (WGA). In contrast, the peptide-protein conjugate NLS-HSA carrying the nuclear localization sequence (NLS) of the SV40 T antigen was only imported but not exported. 3T3 polykaryons were injected with IAF-p53 and photobleached by Scanning Microphotolysis in such a manner that only a single nucleus per polykaryon remained non-bleached. IAF-p53 was found to migrate rapidly (halftime similar to 10 min) from non-bleached into bleached nuclei, while NLS-HSA did not. In digitonin permeabilized cells IAF-p53 was imported into nuclei. When removed from the medium after nuclear accumulation IAF-p53 was exported from the nuclei. Nuclear import and export of IAF-p53 both were rapid (halftimes of a few minutes, 22 degrees C) and strongly inhibited by WGA or incubation on ice. NLS-HSA was only imported but not exported. We conclude that the nucleocytoplasmic transport of p53, in contrast to that of NLS-HSA, is bidirectional and that transport in both directions is carrier mediated and energy dependent. These results suggest that p53 contains nuclear export signals (NES) in addition to import signals (NLS) and thus open new views on the potential regulation of p53 cellular fractions.