title: Regulation of nuclear import and export of negative cofactor 2.
abstract: The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2alpha and NC2beta, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2alpha and NC2beta depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin alpha/beta heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2beta. In contrast, NC2alpha does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2alpha masks the NES in NC2beta, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin alpha/beta-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits.
authors.affiliation: Institute of Biochemistry and Molecular Cell Biology, Department of Molecular Biology, Georg-August-University, Humboldtalle 23, 37073 Göttingen, Germany.
authors.forename: Joerg
authors.initials: J
authors.lastname: Kahle
authors.forename: Elisa
authors.initials: E
authors.lastname: Piaia
authors.forename: Sonja
authors.initials: S
authors.lastname: Neimanis
authors.forename: Michael
authors.initials: M
authors.lastname: Meisterernst
authors.forename: Detlef
authors.initials: D
authors.lastname: Doenecke
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D002460:Cell Line; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
chemical_list: D005231:Fatty Acids, Unsaturated; D028884:Karyopherins; D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D014157:Transcription Factors; D000097606:Exportin 1 Protein; C099112:DRAP1 protein, human; C075995:down-regulator of transcription 1; C038753:leptomycin B