Nuclear localisation signals (NLSs) have been classified as either mono- or bipartite. Genetic analysis and GFP fusions show that the NLS of a Zn-binuclear cluster transcriptional activator of Aspergillus nidulans (PrnA) is tripartite. This NLS comprises two amino-terminal basic sequences and the first basic sequence of the Zn-cluster. Neither the two amino-terminal basic sequences nor the paradigmatic nucleoplasmin bipartite NLS drive our construction to the nucleus. Cryosensitive mutations in the second basic sequence are suppressed by mutations that restore the basicity of the domain. The integrity of the Zn-cluster is not necessary for nuclear localisation. A tandem repetition of the two basic amino-terminal sequences results in a strong NLS. Complete nuclear localisation is observed when the whole DNA-binding domain, including the putative dimerisation element, is included in the construction. At variance with what is seen with tandem NLSs, all fluorescence here is intra-nuclear. This suggests that retention and nuclear entry are functionally 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 proline induction). In contrast with what has been observed by others in A. nidulans, 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.
Copyright 2000 Academic Press.