The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited.
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.
Middeler
G
G
Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, Münster, Germany.
Zerf
K
K
Jenovai
S
S
Thulig
A
A
Tschödrich-Rotter
M
M
Kubitscheck
U
U
Peters
R
R
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D009687:Nuclear Proteins / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000494:pharmacology*
D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D014909:Wheat Germ Agglutinins