The analysis of the transcriptional activator PrnA reveals a tripartite nuclear localisation sequence

Nuclear localisation signals (NLSs) have been classified as either monoor b ipartite. Genetic analysis and GFP fusions show that the NLS of a Zn-binucl ear cluster transcriptional activator of Aspergillus nidulans (PrnA) is tri partite. This NLS comprises two amino-terminal basic sequences and the firs t basic sequence of the Zn-cluster. Neither the two amino-terminal basic se quences nor the paradigmatic nucleoplasmin bipartite NLS drive our construc tion to the nucleus. Cryosensitive mutations in the second basic sequence a re suppressed by mutations that restore the basicity of the domain. The int egrity of the Zn-cluster is not necessary for nuclear localisation. A tande m repetition of the two basic amino-terminal sequences results in a strong NLS. Complete nuclear localisation is observed when the whole DNA-binding d omain, including the putative dimerisation element, is included in the cons truction. At variance with what is seen with tandem NLSs, all fluorescence here is intranuclear. This suggests that retention and nuclear entry are fu nctionally different. With the whole PrnA protein, we observe localisation, retention and also a striking sub-localisation within the nucleus. Nuclear localisation and sub-localisation are constitutive (not dependent on proli ne induction). In contrast with what has been observed by others in A, nidu lans, none of our constructions are delocalised during mitosis. This is the first analysis of the NLS of a Zn-binuclear cluster protein and the first characterisation of a tripartite NLS. (C) 2000 Academic Press.