Import of human and Thermoplasma 20S proteasomes into nuclei of HeLa cells requires functional NLS sequences.
Proteasomes are present both in the nucleus and cytoplasm of eukaryotic cells. Their localization is regulated and changes during the cell cycle. Nuclear localization signal (NLS) type sequences were identified in proteasomes from various organisms. In addition, acidic complementary sequences were identified (cNLS) which could interact with the positively charged NLS, masking or unmasking them and thereby modulating nuclear import. In this paper we show that fluorescently labeled human erythrocyte 20S proteasomes accumulate in the nucleus of digitonin-permeabilized cells. This translocation is ATP-dependent and occurs through the nuclear pore complex as is shown by blocking of the nuclear pores with wheat germ agglutinin. In addition, we used 20S proteasomes from Thermoplasma acidophilum as a model system. Recombinant 20S proteasomes from the archaebacterium Thermoplasma acidophilum are imported into nuclei of HeLa and 3T3 cells similar to their eukaryotic counterpart. We constructed mutants in the putative NLS and cNLS region to study their effect on import. The NLS mutant was not imported into nuclei and showed cytoplasmic staining only. This indicates that this sequence is indeed responsible for nuclear targeting. Mutational studies of the cNLS do not support the involvement of this sequence in regulation of nuclear transport.
Wang
H R
HR
Max Planck Institute for Biochemistry, Martinsried/Germany.
Kania
M
M
Baumeister
W
W
Nederlof
P M
PM
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D009685:Nuclear Envelope / Q000378:metabolism; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex