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.
Greve
Krestine
K
Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, 1353 Copenhagen K, Denmark.
La Cour
Tanja
T
Jensen
Michael K
MK
Poulsen
Flemming M
FM
Skriver
Karen
K
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
C474015:ANAC protein, Arabidopsis; C517610:ATAF1 protein, Arabidopsis; D029681:Arabidopsis Proteins; C107294:CUC2 protein, Arabidopsis; D002352:Carrier Proteins; C099172:NAM protein, Petunia hybrida; D010940:Plant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C474016:RHA2a protein, Arabidopsis; C474017:RHG1a protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases