Article Figures & Data
Figures
- Figure 1.
Protein alignment (left) and unrooted phylogenetic dendrogram (right) of the AtBPM family, CeMEL26 (NP_492449), and HsSpop (AAH03385). Underlined in black is the MATH domain, whereas light gray indicates the position of the BTB/POZ domain. Identical residues are in black and conserved amino acid residues are in gray, respectively. Line in the lower right of the dendrogram gives the distance between each branch (percentage divergence/100). Graphics were done on the Workbench Web site (http://workbench.sdsc.edu) using ClustalW, PHYLIP, and Boxshade version 3.3.1 programs. Trees were generated using the neighbor-joining method (Saitou and Nei, 1987); protein grouping was verified by bootstrapping (1,000 replicates, 111 random seed number; Felsenstein, 1985).
- Figure 2.
Interaction studies of AtCUL3a, AtCUL3b, AtCUL4, and selected AtBPM proteins. A, AtCUL3a and AtCUL3b can both interact in Y2H assays with AtBPM1 and AtBPM3 but not with AtBPM5 and AtBPM6. AtCUL4 was used only with AtBPM1 and did not show any interaction. B, Y2H interaction studies show that AtBPM1 and AtBPM3 assemble to homodimers and heterodimers with the other tested AtBPM proteins. C, Results from the Y2H studies were confirmed for AtCUL3a, AtCUL3b, AtCUL4, and AtBPM1 by pull-down assays with bacterially expressed and purified GST or GST∷AtBPM1 proteins. First lane of each assay shows 1 μL of [35S]Met-labeled protein used for pull downs. SDII, Selection medium for transformation with bait (pBTM112-D9) and prey (pACT2) plasmids supplemented with uracil and His; SDIV, selection medium for interaction studies without uracil and His supplements. Photos were taken from single spots.
- Figure 3.
AtCUL3 proteins interact with AtRBX1 and can be modified by RUB1. A, In Y2H assays, AtRBX1 shows interaction with both AtCUL3 proteins. B, Interaction was confirmed for AtCUL3a by using purified GST or GST∷AtRBX1 protein in pull-down assays. Most outer lane shows 1 μL of in vitro translated AtCUL3a protein used for pull downs. C, Left half, GST∷RUB1 modification of AtCUL3a and AtCUL3b (indicated by arrows); right half, mutagenesis of a conserved Lys to Met at position 678 (AtCUL3K678M) resulted in loss of AtCUL3 modification by GST∷RUB1.
- Figure 4.
Mutagenesis of conserved amino acid residues in AtCUL3a affects interaction with AtBPM proteins. A, Schematic overview of AtCUL3a and locations of mutagenized amino acid residues (underlined and pointed out by arrows). B, Changing two conserved residues at the NH2 terminus, a Ser-50 and a Phe-51 to Ala, disrupted interaction with AtBPM1 but not with AtRBX1. By contrast, mutagenesis of Lys-678 to Met had no influence on AtCUL3a-AtBPM1 assembly.
- Figure 5.
Mutagenesis of conserved residues in AtBPM1 affects assembly with AtCUL3 and AtBPM1 proteins. A, Schematic overview of AtBPM1 and locations of mutagenized amino acid residues (underlined and pointed out by arrows). B, In contrast with AtBPM1 and AtBPM1D204N, AtBPM1L189Stop and AtBPM1D204A did not assemble with AtCUL3a and AtBPM1 proteins in Y2H assays. In addition, AtBPM1D328A and AtBPM1Y330A strongly reduced interaction with AtCUL3a and AtBPM1. The introduction of a point mutation at position 332 (AtBPM1L332A) only affected interaction with AtBPM1. C, Results from the Y2H studies were verified for AtBPM1D204A and AtBPM1L189STOP by pull-down assays with in vitro translated AtCUL3a, AtBPM1 and mutated AtBPM1 versions, and bacterially expressed and purified GST, GST∷AtBPM1, GST∷AtBPM1D204A, or AtBPM1L189STOP fusion proteins.
- Figure 6.
Expression pattern of AtCUL3a, AtCUL3b, and the four tested AtBPM genes. A, Semiquantitative RT-PCR shows expression of all tested genes in root, leaf, shoot, and flower. B to I, Analysis of AtCUL3a promoter GUS lines showed GUS expression in all organs (B, seedling; C, close up to vascular tissue; D and E, lateral roots; F–I, inflorescence). J and K, GUS expression was confirmed for flower tissue by in situ hybridization. Expression was detectable in pistil and anthers (J, sense; K, antisense). L to Q, Analyses of AtCUL3b promoter GUS lines showed expression similar to pAtCUL3a∷GUS lines in all tested tissues (L, root; M, seedling; N, rosette leafs; O, leaf close up; P, inflorescence with two cauline leafs; Q, flower).
- Figure 7.
Schematic model of AtBPM and AtCUL3 assembly. A, AtBPM proteins assemble with other AtBPM proteins by using their BTB/POZ domain (black bar). B, AtCUL3 proteins interact at their NH2-terminal part with the BTB/POZ domain of AtBPM proteins and within their COOH-terminal region with AtRBX1. The AtCUL3 proteins can be modified by the ubiquitin-related protein RUB1. As potential substrate adaptors, the AtBPM proteins contact substrates via their MATH domains (white bar).
