Numerous, highly conserved RING-H2 domains are found in the model plant Arabidopsis thaliana (thale cress). To characterize potential RING-H2 protein interactions, the small RING-H2 protein RHA2a was used as bait in a yeast two-hybrid screen. RHA2a interacted with one of the plant-specific NAC [NAM ('no apical meristem'), ATAF1/2, CUC2 ('cup-shaped cotyledons 2')] transcription factors, here named ANAC (abscisic acid-responsive NAC). The core RING-H2 domain was sufficient for the interaction. The ability of 11 structurally diverse RING-H2 domains to interact with ANAC was then examined. Robust interaction was detected for three of the domains, suggesting multi-specificity for the interaction. The domains that interacted with ANAC contain a glutamic acid residue in a position corresponding to a proline in many RING-H2 domains. Conversion of this glutamic acid residue into proline in RHA2a decreased its ability to interact with ANAC, most likely by changing the interaction surface. This suggested that a short, divergent region in RING-H2 domains modulate interaction specificity. ANAC contains a degenerate bipartite nuclear localization signal (NLS), while RHG1a, also identified as an ANAC interaction partner, contains a basic NLS. Both signals localized beta-glucuronidase reporter fusions to the nucleus. N-terminally truncated RHA2a also directed nuclear localization, apparently dependent on basic amino acids in the RING-H2 domain. Nuclear co-localization of the RING-H2 proteins and ANAC may enable their interaction in vivo to regulate the activity of the ANAC transcription factor.
|Issue number||Pt 1|
|Number of pages||12|
|Publication status||Published - 2003|
- Abscisic acid-responsive nitrogen assimilation control protein (NAC)
- Cellular localization signal
- Plant model
- RING-H2 target protein
- Ubiquitination pathway
Greve, K., La Cour, T., Jensen, M. K., Poulsen, F. M., & Skriver, K. (2003). Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization. Biochemical Journal, 371(Pt 1), 97-108.