Volume 5, Issue 4, Pages 876-888 (July 2012) Studies on Differential Nuclear Translocation Mechanism and Assembly of the Three Subunits of the Arabidopsis thaliana Transcription Factor NF-Y  Dieter Hackenberg, Yanfang Wu, Andrea Voigt, Robert Adams, Peter Schramm, Bernhard Grimm  Molecular Plant  Volume 5, Issue 4, Pages 876-888 (July 2012) DOI: 10.1093/mp/ssr107 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Detection of the Subcellular Localization of NF-Y Proteins in Arabidopsis Leaves. NF-Y–eGFP-gene fusions under the control of a 35S::CaMV promoter were used for transient transformation via particle bombardment. The GFP fluorescence was detected by confocal laser scanning microscopy (left row). Location of the nucleus and cell walls was identified after DAPI staining and bright field microscopy, respectively (central rows). Whereas NF-YA2 (A) and NF-YC9 (C) were nuclear-located, NF-YB10 (B) is distributed in the cytoplasm. NF-YB10 is translocated to the nucleus by co-expression of NF-YC2 (D). The right row displays the merge of green fluorescence and the DAPI stain. Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Detection of Protein–Protein Interaction among NF-YB10 and NF-YC9 In Vitro and In Vivo. [35S]-methionine-labeled NF-YB10 can be pulled down from a GST::NF-YC9 matrix (A) (right lane) but not from a GST matrix (left lane). The interaction among NF-YB10 and NF-YC9 can be detected in the yeast two-hybrid system by the activation of the reporter genes HIS3 and lacZ, which lead to an improved growth on SC-HLTU media (B) (upper panel) and increased β-galactosidase activity (lower panel) compared to the control experiment (expression of NF-YB10 and an empty prey vector). Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 BiFC Visualization of Arabidopsis NF-YB10-NF-YC9 and NF-YB3-NF-YC9 Dimers in Nicotiana benthamiana Leaf Epidermis Cells after Agrobacterium-Mediated Transient Transformation. For specification of the specific bimolecular interaction of the NF-Y subunits in the nucleus, endoplasmic reticulum that also surrounds the nucleus was visualized by co-transformation of a specific CFP-fused ER-marker (ABRC stock: CD3-953). Detection of the YFP (left row), CFP (second row from the left side), merge of YFP and CFP fluorescence (second row from the right side), and bright field microscopy (right row) of NF-YC9_YFPN/NF-YB10_YFPC/CD3-953_CFP (A), NF-Y B10_YFPN/NF-Y C9_YFPC/CD3-953_CFP (B), NF-YC9_YFPN/NF-YB3_YFPC/CD3-953_CFP (C), and NF-Y B3_YFPN/NF-Y C9_YFPC/CD3-953_CFP (D) are displayed. NF-YB3 as well as NF-YB10 interact with NF-YC9. The resulting dimer formation is localized inside the nucleus. Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Overview about the Bilateral Interactions among Two NF-Y Proteins Tested in Yeast Two-Hybrid Experiments. The mean β-galactosidase activities of three biological replica of diploid yeast cells possessing bait and prey constructs for the expression of singular NF-Y proteins were plotted as a color-coded diagram. The used bait constructs are positioned on the x-axis and the prey constructs on the y-axis. Successful protein–protein interactions were plotted as heat color-encoded mean values of all three biological replicas exceeding 0.2 relative β-galactosidase activity units. Interaction among two NF-Y proteins, which were only detectable in one of both reciprocal bait/prey combinations were plotted as different intense gray shades of color. Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Sequence Alignments among the Highly Conserved Domains of the Arabidopsis thaliana, Saccharomyces cerevisiae, and Homo sapiens NF-Y Subunits. Functional domains are labeled in black boxes above the sequence alignment of NF-YA (A), NF-YB (B), and NF-YC (C). Conserved cysteine residues of NF-YB subunits are labeled as C1, C2, and C3 (red) and putative H-bonds between arginine and aspartate are labeled with two black arrows (Romier et al., 2003). Amino acid residues highlighted yellow are strictly conserved in all representatives of the corresponding subunit family, turquoise-labeled amino acids are identical at those positions, and green-labeled amino acid residues possess side chains with similar biochemical properties. Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Proof of the Detectable Interaction among NF-YA and NF-YB using the Yeast Two-Hybrid System. Diploid yeast cells possessing NF-YA and NF-YB as bait and prey constructs did not show any activation of the yeast two-hybrid reporter genes HIS3 and lacZ. Co-transformation of these diploid yeast cells with a gene encoding a NF-YC subunit that was fused neither to a Gal4-AD nor a LexA-BD of the yeast two-hybrid system enables the corresponding yeast cells to grow on SC-HLTU media (A) and exhibit significantly increased β-galactosidase activity (B). Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Models of the Three-Dimensional Structure of the Human NF-YB as well as Arabidopsis NF-YB10 on the Base of the Crystal Structure of the Human NF-YB/C Heterodimer (PDB ID 1N1J; Romier et al., 2003). Peptide chains are displayed as flat ribbons and cysteine side chains as ball-and-stick models with red-labeled sulfur atoms. The human NF-YB peptide chain (A) exhibits three cysteine residues. The spatial orientation of C1 and C2 would allow an intramolecular disulfide bond. The Arabidopsis NF-YB10 peptide chain (B) possesses only cysteines C2 and C3, which do not allow the formation of an intramolecular disulfide bond. (C) Identification of protein–protein interactions among NF-YB and C under different oxidizing conditions using the yeast two-hybrid system. Determination of the β-galactosidase activity of diploid yeast cells possessing yeast two-hybrid bait and prey constructs for the expression of Arabidopsis or human NF-YB and NF-YC. Heterodimerization of the human NF-YB and NF-YC is inhibited by treatment with more than 500 μM H2O2, whereas Arabidopsis NF-YB10 and NF-YC2 can form heterodimers even if 5 mM H2O2 were added. Molecular Plant 2012 5, 876-888DOI: (10.1093/mp/ssr107) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions