Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

Abstract

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.

BASIC PENTACYSTEINE Proteins Mediate MADS Domain Complex Binding to the DNA for Tissue-Specific Expression of Target Genes in Arabidopsis

“…By contrast, plants that contained the reporter construct in which five C-boxes were mutated displayed ectopic expression in the carpel and stamens, suggesting that the number of BPC binding sites is important for correct gene expression. Our yeast interaction studies showed that BPC proteins of class I can interact with each other, and the fact that those of class II also interact between them and with those of class I (Wanke et al, 2011) further supports the idea that multiple DNA interactions combined with BPC protein-protein interactions will induce conformational changes into the STK promoter region, also corroborated by the previous reported in vitro TPM analysis (Kooiker et al, 2005).…”

Degradation of Arabidopsis CRY2 Is Regulated by SPA Proteins and Phytochrome A

“…Expression of full-length fusion proteins was verified by immunoblot analysis (see Supplemental Figure 9 online). As shown in Figure 5, cry2, CIB1, and all four SPAs localized to nuclei as additionally verified using the mCherry-NLS reporter (Wanke et al, 2011). Moreover, the pictures from the merged GFP and RFP channels showed that cry2 colocalizes with CIB1 and all four SPAs.…”

“…Afterwards some plant material was harvested followed by protein extraction for immunoblot analysis. In vivo interaction studies of the full-length fusion proteins were measured by FRET-FLIM with a confocal stage scanning microscope according to Wanke et al (2011). For each tested combination of fusion proteins, three measurements of fluorescence lifetime decays were recorded and mean values estimated.…”

Light-Activated Phytochrome A and B Interact with Members of the SPA Family to Promote Photomorphogenesis in Arabidopsis by Reorganizing the COP1/SPA Complex

“…All FLIM measurements were performed as previously described (Wanke et al, 2011), with the following modifications for CFP-YFP FRET. A pulsed 440-nm diode laser (Picoquant LDH-D-C-440), operating at a repetition rate of 20 MHz, was used for excitation, in conjunction with LD01-439/8-12.5 (Semrock) cleanup interference filters.…”