The basic domain in the bZIP regulatory protein Opaque2 serves two independent functions: DNA binding and nuclear localization.

For organisms to grow and develop, transcriptional regulatory proteins must localize to the nucleus. This movement is mediated by nuclear localization sequences (NLSs) which consist of short stretches of basic amino acids that are part of the structure of mature nuclear proteins. Two NLSs have been previously identified in the maize regulatory protein Opaque2 (O2), a member of the basic-domain, leucine-zipper (bZIP) class of proteins. In this report, it has been determined that both of these NLSs are necessary for import of O2, and the focus has been on a functional analysis of the more efficient, bipartite NLS that is present in the basic or DNA-binding domain. A mutation which contains altered amino acids on both parts of this NLS severely reduced nuclear targeting, and led to the definition of two classes of bipartite NLSs. Since the bipartite NLS is located in the highly conserved. DNA-binding domain, the dual role of this domain was examined using the maize mutant o2-676, in which a conservative mutation eliminates the DNA-binding function. The o2-676 protein localized to the nucleus in maize and the bZIP domain from the mutant protein was sufficient to redirect a reporter protein to the nucleus in transgenic plant cells. Thus, it was possible to show that the nuclear targeting function of this domain is independent of DNA binding. Sequence conservation of the basic domain of other bZIP proteins suggests that the bifunctionality of this domain may be conserved in all members of this class; a consensus sequence for a bipartite NLS in bZIP proteins is suggested.