Fig. 2. Stoichiometric incorporation of GLMN into…
Fig. 2. Stoichiometric incorporation of GLMN into numerous SCF assemblies via RBX1’s RING domain
A.…
Fig. 2. Stoichiometric incorporation of GLMN into numerous SCF assemblies via RBX1’s RING domainA. Gel filtration chromatography elution profiles of GLMN, RBX1-CUL1, and CAND1 alone (black, red, and green, respectively), mixtures of RBX1-CUL1 and CAND1 (purple), and RBX1-CUL1 with CAND1 and GLMN (orange). RBX1-CUL1 corresponds to the “split ‘n coexpress” version in which full-length CUL1 is generated by coexpression of the N- and C-terminal domains as two noncovalently-associated polypeptides that migrate closely on SDS-PAGE (Zheng et al., 2002b). Sypro-stained SDS-PAGE gels of fractions from gel filtration peaks are shown below chromatograms. B. Gel filtration chromatography elution profiles and Sypro-stained SDS-PAGE gels of fractions for GLMN and RBX1-CUL1~NEDD8 alone (black and blue, respectively), and of a mixture of RBX1-CUL1~NEDD8 and GLMN (red) using “split ‘n coexpress” RBX1-CUL1. These experiments were performed at the same time as those in panel A, with the same data for GLMN shown in both panels for reference. C. Gel filtration chromatography elution profiles and Sypro-stained SDS-PAGE gels of fractions for GLMN, RBX1-CUL1 C-terminal domain (CTD), and a version lacking the RING domain (RBX1ΔRING) alone (black, red, and blue, respectively), and mixtures of RBX1-CUL1CTD and RBX1ΔRING-CUL1CTD with GLMN (green and orange, respectively). D. Anti-biotin western blots showing polyubiquitination time-courses of a biotinylated CyclinE phosphopeptide in reactions containing 500 nM CDC34, 200 nM SCFFBW7, and the indicated ratio of GLMN:SCF (1× = 200 nM GLMN). E. Anti-p27 western blots showing time-courses of SCFSKP2-CKSHS1-mediated polyubiquitination of phospho-p27, in the presence of 3x GLMN:SCF, or with GLMN and in competition assays also containing the indicated ratios of the isolated RBX1 RING domain:SCF. F. Anti-biotin western blots showing time-courses of SCFFBW7-mediated polyubiquitination of a biotinylated CyclinE phosphopeptide, in the presence of 3x GLMN:SCF and in competition assays containing the indicated ratios of the isolated RBX1 RING domain:GLMN. G. Quantification of dissociation constant (mean ± SD) from FRET-based binding assay between GLMN-YFP and CFP-RBX1-CUL1CTD, CFP-RBX1-CUL1CTD~NEDD8, or CFP-RBX1RING. Error bars represent standard error from 3 independent experiments (raw data shown in Fig. S3). H. Structural models of GLMN-RBX1-CUL1, GLMN-RBX1-CUL1-CAND1, and GLMN-RBX1-NEDD8~CUL1-SKP1-FBW7-CyEphosphopeptide based on superimposing GLMN-RBX1 (RING) on the RBX1 RING domain of the prior RBX1-CUL1 (Zheng et al., 2002b) and RBX1-CUL1-CAND1 (Goldenberg et al., 2004) structures, and on the structural model of RBX1-CUL1~NEDD8 (Duda et al., 2008) also modeled with SKP1-FBW7- CyEphosphopeptide docked onto the N-terminal domain of CUL1 (Hao et al., 2007).
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