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The terminal domain of AGL15 contains two conserved motifs, one of which is a putative repression motif
Comparison of amino acid sequences of class-II ERF repressors revealed the conservation of the sequence motif L /FDLN L /F(x)P, designated as the ERF-associated amphiphilic repression (EAR) motif, and mutational analysis demonstrated that this motif is essential for repression (Ohta et al., 2001). A similar motif was also identified in a number of zinc-finger proteins from a variety of species (Ohta et al., 2001), and in some Aux/IAA proteins (Tiwari et al., 2004) that function as repressors. Here we demonstrate that the repressive capacity of AGL15 was dependent on the presence of the C-terminal domain containing the LxLxL motif (Figure 3c), and that the LxLxL motif was required for the interaction of AGL15 with SAP18 (Figure 4). AtSAP18 has also been shown to interact with ERF3 and ERF4 (Song and Galbraith, 2006), which contain the motifs FQFDLNFPP and LDLDLNLPP, respectively.
SAP18 did not interact with any of the other MADS-domain proteins we tested, which is unsurprising given that the interaction occurs at least in part via the divergent C-terminal domain and not only via the highly conserved MADS or moderately conserved I and K domains. However, SAP18 did not interact, at least in yeast, with the closest relative of AGL15, AGL18, which also contains a similar LxLxL motif (DTSLQLGLSS) and exhibits functional redundancy in planta with AGL15 (Adamczyk et al., 2007). However, no AGL18-interacting partners have been demonstrated using the yeast system in our lab, or have been reported in the literature, even though such experiments have been performed (de Folter et al., 2005; Verelst et al., 2007). The literature contains a number of examples where a full-length MADS-domain protein, when fused to the GAL4-DBD, is unable to interact with partners with which truncated forms do interact (Fujita et al., 2003; Yang and Jack, 2004; Yang et al., 2003a,b). However, a truncated form of AGL18 lacking the MADS domain also failed to interact with any of the AGL15-interacting proteins (K. Hill, unpublished data). There are examples where an interaction shown to occur between full-length MADS in planta does not occur in the yeast system (Immink et al., 2002), and could reflect a conformation effect brought about by the GAL4-DBD fusion masking some interactions. SAP18 also did not interact with a form of SVP that lacks the MADS domain but has an LxLxL motif within the C-terminal domain (data not shown). This may reflect the involvement of the K domain in mediation of the AGL15–SAP18 interaction that is only weakly conserved in primary structure between MADS MIKC proteins. The last member of the Arabidopsis MADS family with a similar LxLxL motif, AGL24, was not tested in the yeast system for interaction with SAP18 because it is not expressed in embryonic tissue (Lehti-Shiu et al., 2005). Because SAP18 is more widely expressed, it is possible that it does interact with AGL24 in other developmental contexts.
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