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 s ynthesized in the form of a membrane-attached precursor molecule. When cell s are deprived of sterols, a two-step proteolytic processing releases the t ranscriptionally active N-terminal segment of SREBP-2, thereby allowing it to enter the nucleus. In previous studies, we showed that the nuclear impor t of SREBP-2 occurs via the direct interaction of importin beta with the HL H-Zip domain. In this study, in order to more completely understand the int racellular 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 tha t 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 moti f disrupted the dimerization. It was also demonstrated that this mutant pro tein 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 appeare d to be composed of a dimeric form of SREBP-2 and importin beta. In additio n, the SREBP-2 binding domain of importin beta corresponded to an overlappi ng but not identical region for importin cu binding, which may explain how importin beta is able to recognize the dimeric HLH-Zip directly. These resu lts indicate that dimerization is a prerequisite process for the nuclear im port of SREBP-2 mediated by importin beta.