Dimerization of sterol regulatory element-binding protein 2 via the helix-loop-helix-leucine zipper domain is a prerequisite for its nuclear localization mediated by importin beta.
The sterol regulatory element-binding protein 2 (SREBP-2), a transcription factor of the basic helix-loop-helix-leucine zipper (bHLH-Zip) family, is synthesized in the form of a membrane-attached precursor molecule. When cells are deprived of sterols, a two-step proteolytic processing releases the transcriptionally active N-terminal segment of SREBP-2, thereby allowing it to enter ... the nucleus. In previous studies, we showed that the nuclear import of SREBP-2 occurs via the direct interaction of importin beta with the HLH-Zip domain. In this study, in order to more completely understand the intracellular dynamics of SREBP-2, we focused on the manner by which importin beta recognizes SREBP-2 at the initial step of the import. It was found that the active form of SREBP-2 exists as a stable dimer in solution and that the substitution of leucine residues for alanine in the leucine zipper motif disrupted the dimerization. It was also demonstrated that this mutant protein did not enter the nucleus either in vivo or in vitro. Solution binding assays, which involved the chemical cross-linking of wild-type or mutated SREBP-2 with importin beta, revealed that the import-active complex appeared to be composed of a dimeric form of SREBP-2 and importin beta. In addition, the SREBP-2 binding domain of importin beta corresponded to an overlapping but not identical region for importin alpha binding, which may explain how importin beta is able to recognize the dimeric HLH-Zip directly. These results indicate that dimerization is a prerequisite process for the nuclear import of SREBP-2 mediated by importin beta.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Base Sequence, Binding Sites, Biological Transport, Active, Carrier Proteins, Cell Nucleus, DNA Primers, DNA-Binding Proteins, Dimerization, Hela Cells, Helix-Loop-Helix Motifs, Humans, Karyopherins, Leucine Zippers, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Localization Signals, Nuclear Proteins, Protein Structure, Quaternary, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sterol Regulatory Element Binding Protein 2, Transcription Factors, alpha Karyopherins
Amino Acid Sequence, Amino Acid Substitution, Base Sequence, Binding Sites, Biological Transport, Active, Carrier Proteins, Cell Nucleus, DNA Primers, DNA-Binding Proteins, Dimerization, Hela Cells, Helix-Loop-Helix Motifs, Humans, Karyopherins, Leucine Zippers, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Localization Signals, Nuclear Proteins, Protein Structure, Quaternary, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sterol Regulatory Element Binding Protein 2, Transcription Factors, alpha Karyopherins
Mol. Cell. Biol.
Date: Apr. 01, 2001
PubMed ID: 11283257
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