Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

BACKGROUND: CrMYC2 is an early jasmonate-responsive bHLH transcription factor involved in the regulation of the expression of the genes of the terpenic indole alkaloid biosynthesis pathway in Catharanthus roseus. In this paper, we identified the amino acid domains necessary for the nuclear targeting of CrMYC2. FINDINGS: We examined the intracellular localization of whole CrMYC2 and of various deletion mutants, all fused with GFP, using a transient expression assay in onion epidermal cells. Sequence analysis of this protein revealed the presence of four putative basic nuclear localization signals (NLS). Assays showed that none of the predicted NLS is active alone. Further functional dissection of CrMYC2 showed that the nuclear targeting of this transcription factor involves the cooperation of three domains located in the C-terminal region of the protein. The first two domains are located at amino acid residues 454-510 and 510-562 and contain basic classical monopartite NLSs; these regions are referred to as NLS3 (KRPRKR) and NLS4 (EAERQRREK), respectively. The third domain, between residues 617 and 652, is rich in basic amino acids that are well conserved in other phylogenetically related bHLH transcription factors. Our data revealed that these three domains are inactive when isolated but act cooperatively to target CrMYC2 to the nucleus. CONCLUSIONS: This study identified three amino acid domains that act in cooperation to target the CrMYC2 transcription factor to the nucleus. Further fine structure/function analysis of these amino acid domains will allow the identification of new NLS domains and will allow the investigation of the related molecular mechanisms involved in the nuclear targeting of the CrMYC2 bHLH transcription factor.