Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization.
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.
Mesh Terms:
Amino Acid Sequence, Arabidopsis Proteins, Carrier Proteins, Cell Nucleus, Cytoplasm, Gene Expression Regulation, Plant, Models, Molecular, Molecular Sequence Data, Plant Proteins, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Repressor Proteins, Substrate Specificity, Transcription Factors, Two-Hybrid System Techniques, Ubiquitin
Amino Acid Sequence, Arabidopsis Proteins, Carrier Proteins, Cell Nucleus, Cytoplasm, Gene Expression Regulation, Plant, Models, Molecular, Molecular Sequence Data, Plant Proteins, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Repressor Proteins, Substrate Specificity, Transcription Factors, Two-Hybrid System Techniques, Ubiquitin
Biochem. J.
Date: Apr. 01, 2003
PubMed ID: 12646039
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