Nuclear and nucleolar targeting of human ribosomal protein S6.
Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6.
Schmidt
C
C
Institut für Physiologische Chemie, Abteilung Molekularbiologie, Universität Hamburg, Germany.
Lipsius
E
E
Kruppa
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002466:Cell Nucleolus / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D006801:Humans; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D001616:beta-Galactosidase