Figure 1.
Protein organization and subcellular localization of GAGA-motif binding proteins.
(A) Schematic representation of Drosophila and plant GAGA-motif binding proteins. Locations of homologous domains are shown for the DNA-binding domains (black), putative protein interaction domains (grey) and Q-rich domains (dark grey). (B) Protein sequence alignment of AtBPC6 orthologs. Conserved amino-acids are highlighted, invariant positions (black) and positions that are preserved in at least half of the aligned sequences (grey). Sequences are retrieved from Olimarabidopsis (Olimarabidopsis pumila), Cardamine (Cardamine pratensis), Solanum (Solanum lycopersicum), Medicago (Medicago truncatula) and Oryza (Oryza sativa). Three distinct domains with putative conserved functions are predicted and highlighted by bars on top of the sequences: Coiled-coil (checked-grey), nuclear localization signal (dark grey) and zinc-finger like DNA-binding domains (black). Invariant Cystein positions within the basic DNA-binding domain are indicated by asterisks. (C) Schematic representation of AtBPC6 fragments used for functional analyses and hybrid protein fusions. (D) Laser confocal microscopy analysis of BPC6-GFP, BPC6-NLS-GFP and mGFP localization in Nicotiana benthamiana epidermis cell nuclei. (E) Hetreologous expression in Nicotiana benthamiana epidermis cells identified the 31 amino-acid long NLS to be responsible for targeting GFP-fusion proteins to the nucleus and the nucleolus.
Figure 2.
Stable localization of BPC6 fusion proteins to the nucleus.
Quantification of fluorescence signals in Nicotiana benthamiana nuclei using flow-cytometry. Nuclei are prepared from non-transformed wildtype cells (A) and epidermis cells expressing BPC6-GFP (B), BPC6-NLS-GFP (C), mGFP (D). GFP fluorescence and DNA content is measured after incubation of nuclei in propidium iodide (PI) solution. Representative signal intensity plots of PI- and GFP-fluorescence for ∼250000 events are shown (left). Histograms of PI-fluorescence (top right) and GFP-fluorescence (bottom right) intensity counts are given for each of the measurements. For comparison, normalized background fluorescence of non-transformed wildtype cells are accompanying histograms of GFP-transformed cells (black lines). BPC6-GFP and BPC6-NLS-GFP are retained inside the nucleus in a stabile manner, while mGFP leaks out and gives background signals perfectly overlapping with wildtype (black line). Black arrowheads indicate significant retention of the GFP signals inside the nuclei.
Figure 3.
Coiled coil domains of Arabidopsis group II BBR/BPC proteins are essential for homotypic dimerization.
Interaction analyses with hybrid fusion proteins of AtBPC6 and its fragments by yeast two-hybrid are shown (A). The BPC6-ΔC fragment containing the coiled-coil domain interacts with full-length BPC6 and BPC6-ΔC in a hybrid-fusion dependent manner. Interaction of group II with group I and II proteins in the yeast two-hybrid system is tested with AtBPC1, AtBPC4 and AtBPC6 hybrid-fusion proteins (B). The coiled-coil domains of group II hybrid proteins BPC4 and BPC6 are essential for homotypic dimer formation. Interaction is tested by growth on adenine-deficient CSM selection media and β-galactosidase reporter activity. Enzymatic activity of the β-galactosidase reporter is quantified from triple measurements of MUG substrate assays and three independent transformations [n = 9]. Statistical background for no significant interaction is calculated from combinations with empty AD-clones and is shown as dotted line. Asterisks mark combinations of hybrid fusion proteins that mediate significantly increased reporter enzyme activities over the background: (**) indicates strong and highly significant (p≤0,0001) interaction; Weak but significant interaction (p≤0,01) is indicated by (*).
Figure 4.
Arabidopsis group II proteins BPC4 and BPC6 form homotypic dimers in planta.
Bi-molecular fluorescence complementation (BiFC) is used for in planta interaction studies. (A–F) Expression of indicated BPC1, BPC4 and BPC6 split-YFP hybrid fusion proteins are examined in transiently transformed Nicotiana benthamiana epidermal cells. The coiled-coil domain is essential for homotypic dimerization of BPC4 and BPC6 in the nucleus and the nucleolus.
Figure 5.
The Alanine zipper coiled-coil forms dimers via electrostatic interaction.
(A) Alanine zipper regions of AtBPC6 and of other proteins are compared to the Leucine zipper of the C-Jun oncogene: Physcomitrella: [GenBank: AB292414], Microcystis: [GenBank: AP009552], Azorhizobium: [GenBank: ABA21837], Aspergillus: [GenBank: XM_750964], Homo: [GenBank: BC146794], Sus: [GenBank: XM_001925969]. Conserved Alanines at the ‘d’ positions of the Alanine zipper are highlighted with black background color. Conserved positions of positively or negatively charged residues are indicated in blue or in red colors, respectively. (B) Helical wheel diagram of an Alanine zipper homodimer. The diagram depicts the clockwise axial rotation of the helices as viewed from their N-termini. The conserved Alanines form a core and are flanked by the positive (blue) and negative (red) charged residues that oppose each other. The wheel starts with Ala41 and ends with Ala76. Ribbon dimer model of the AtBPC6 Alanine zipper (C) and the C-Jun Leucine zipper regions (D). Monomers are either shown in green or in yellow. Conserved alanines or leucines in the core of the respective dimer are displayed as ball structures. The Leucines of the C-Jun dimer are involved in binding and form a hydrophobic core, which cannot be seen in the AtBPC6 Alanine zipper. Enlarged side (E) and top (F) view of the Alanine zipper homology model. Positively (blue) and negatively (red) charged side chains embrace the alanines (green) and form salt bridges or hydrogen bonds (turquoise). Schematic overview of the residues that support the stable dimer structure of the homology modeled AtBPC6 Alanine zipper via salt bridges (G) or hydrogen bonds (H). Amino acids with positive or negative charges are given in blue or red, respectively. Schematic drawing of the BBR/BPC group II monomers and dimers (I). The so far characterized domains imply a functional model, in which the proteins form homotypic dimers within group II in a parallel manner. As a consequence, their two DNA-binding domains make contact to neighboring GAGA-motifs on the same side of the strand.
Figure 6.
AtBPC6 forms parallel dimers in planta.
(A–C) Confocal laser scanning microscopy analysis of GFP-/RFP-fusion proteins and mCherry-NLS in transiently co-transformed Nicotiana benthamiana epidermis cells. All proteins localize to the nucleus. GFP fluorescence intensities (D) and RFP-FRET fluorescence intensities (E) under GFP-excitation light. (F) In vivo GFP fluorescence life time measurements of all four possible GFP/RFP protein combinations fused to either the N- or the C-terminus of AtBPC6. BPC6-GFP and BPC6-GFP/mCherry-NLS combinations serve as controls. Pictographs (right hand side) display the only possible zipper orientations that are in accordance with the GFP fluorescence life time measurements.