Functional analysis of eukaryotic 20S proteasome nuclear localization signal.
The 20S proteasome is widely viewed at as a cytoplasmic multicatalytic proteinase complex: immunocytochemical investigations, however, show that proteasomes are localized in the cytoplasm as well as in the nucleus within the same cell. Strong nuclear accumulation of proteasomes is observed in rapidly dividing cells such as in the early stages of Drosophila embryogenesis and in tumorigenic cells. In fact, dependent on the metabolic state of a certain tissue or cell type its cellular distribution appears differentially regulated. Several of the proteasomal alpha-type subunits carry putative nuclear localization signals which may or may not take part in the regulation of the intracellular distribution of 20S proteasomes. We have examined the functional role of the putative nuclear localization signal (NLS) -KKKQKK-in the Drosophila PROS-28.1 subunit by deletion mutagenesis and transfection experiments. Linkage of the putative PROS-28.1 NLS to BSA as reporter protein and in vitro import studies with permeabilized mouse NIH 3T3 cells show that this NLS is able to induce complete translocation of the reporter protein into the cell nucleus. For analysis of the NLS within the 28-kDa subunit, cDNA deletion constructs were cloned into a pSG5 expression vector and transiently transfected into mouse fibroblast cells. Whereas the deletion of the NLS alone resulted only in a slight impairment of subunit transport into the nucleus, removal of the C-terminal 96 amino acid residues abolished nuclear translocation completely.
Knuehl
C
C
Institut für Biochemie, Charité, Hunboldt Universitat zu Berlin, Federal Republic of Germany.
Seelig
A
A
Brecht
B
B
Henklein
P
P
Kloetzel
P M
PM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D004330:Drosophila; D005057:Eukaryotic Cells; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000276:immunology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021382:Protein Sorting Signals / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism
D000906:Antibodies; D009097:Multienzyme Complexes; D021382:Protein Sorting Signals; D012710:Serum Albumin, Bovine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex