Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro.
Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits. We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (Hsc iota, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct non-nuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both cell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence.
Nederlof
P M
PM
Department of Structural Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Wang
H R
HR
Baumeister
W
W
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010798:Phycocyanin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D012710:Serum Albumin, Bovine / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013822:Thermoplasma / Q000201:enzymology*
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010446:Peptide Fragments; C002497:allophycocyanin; D010798:Phycocyanin; D012710:Serum Albumin, Bovine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex