Middeler G, Zerf K, Jenovai S, Thulig A, Tschödrich-Rotter M, Kubitscheck U, Peters R
Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, Münster, Germany.
Oncogene. 1997 Mar 27;14(12):1407-17. doi: 10.1038/sj.onc.1200949.
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 photo-bleached 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 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 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.
人p53在大肠杆菌中表达、纯化，用碘乙酰胺荧光素（IAF）标记，并对其序列特异性DNA结合和表位分布进行表征。将IAF-p53注射到3T3细胞的细胞质或细胞核中，几分钟内它就会进入细胞核或从细胞核中输出。同时注射凝集素麦胚凝集素（WGA）可抑制其进入。相比之下，携带SV40 T抗原核定位序列（NLS）的肽-蛋白质偶联物NLS-HSA仅能进入细胞核而不能输出。向3T3多核体细胞注射IAF-p53，并通过扫描微光解进行光漂白，使得每个多核体细胞中仅剩下一个未漂白的细胞核。结果发现IAF-p53能迅速（半衰期10分钟）从未漂白的细胞核迁移到漂白的细胞核中，而NLS-HSA则不能。在用洋地黄皂苷通透处理的细胞中，IAF-p53能进入细胞核。在核积累后从培养基中去除时，IAF-p53会从细胞核中输出。IAF-p53的核输入和输出都很快（在22℃下半衰期为几分钟），并且受到WGA或在冰上孵育的强烈抑制。NLS-HSA仅能进入细胞核而不能输出。我们得出结论，与NLS-HSA相比，p53的核质运输是双向的，并且两个方向的运输都是载体介导的且依赖能量。这些结果表明，p53除了有输入信号（NLS）外还含有核输出信号（NES），因此为p53细胞组分的潜在调控开辟了新的视角。
