Figure 1. Cdt1 is a novel MT-binding... | Rockefeller University Press

Figure 1.

$Figure 1. Cdt1 is a novel MT-binding protein. (A) Disorder prediction in human Cdt1. Disordered prediction (sum of 10 programs) is plotted as a function of residue number. (B) Seven homology models of full-length Cdt1; colored from N terminus (blue) to C terminus (red) with regions of low confidence or low overlap shown in translucent. Conserved WHM and WHC domains shown and aligned in gray and black, respectively, for clarity. Magnified is the PyMol rendered cartoon representation showing superimposition of the C-terminal human Cdt1 (410–546 aa, in red) generated using Phyre2 server with available NMR structure (PDB ID: 2KLO; in green) of mouse C-terminal Cdt1 (420–557 aa). (C) Constructs used in this study, full-length Cdt1 (1–546 aa), its deletion variant devoid of N-terminal 92 aa (92–546), and N- and C-terminal deletion variants generated in fusion with an N-terminal GFP tag for expression in bacteria are shown as black bars. Disordered domain shown in blue, WHM domain shown in yellow, and WHC domain shown in red. (D) SDS-PAGE (15%) of the indicated purified recombinant proteins along with native tubulin purified from porcine brain. M, molecular mass standard; *, degradation bands (confirmed by Western blot) in Cdt192–410. Arrows point toward the band corresponding to the protein of interest. (E) Western blots showing MT cosedimentation for Cdt192–546 (GFP-tagged) and full-length Cdt11–546 (without any tag), 1 µM each; purified from bacteria with the indicated concentrations of taxol-stabilized MTs (in μM). Samples fractionated as supernatant (S) and pellet (P) were analyzed by Western blot and probed with antibodies against 6×-His tag or anti-Cdt1 as indicated in each case to detect Cdt1. Ponceau S–stained tubulin. (F) Quantification (mean ± SD, n = 3) of the Cdt192–546 and Cdt11–546 blots using ImageJ. The 95% CI values obtained for the fit were 0.60–0.94 for Bmax, 0.37–4.04 for Kd, and 0.07–0.29 for the background for the former construct; and 0.63–0.79 for Bmax, 1.33–3.25 for Kd, and 0.08–0.18 for the background for the latter construct. (G) Representative Western blots of the binding of indicated deletion fragments to MTs in a cosedimentation assay with the indicated MT concentrations (top). (H) Quantification of binding (mean ± SD, n = 3) of the deletion fragments to MTs. (I) Quantification of MT-binding of Cdt1’s smallest C-terminal fragment, Cdt1410-546, and a representative Western blot (top) from three independent experiments (mean ± SD, n = 3). (J) Blot overlay assay to study Cdt1–Hec1 interaction. Indicated proteins (0.5 µg each) were loaded as bait on 18% SDS-PAGE gel, transferred to nitrocellulose membrane, and blocked with 5% SM-TBST. Hec1::Nuf2-His dimeric complex (1 µg; a generous gift from A. Desai and D. Cheerambathur, University of California, San Diego, La Jolla, CA) was overlaid as prey protein on the membrane for 12 h at 4°C. The blot was washed and probed with anti-Hec1 antibody (1:2,000; 9G3; Abcam) followed by chemiluminescence. The same blot was probed with Ponceau S.$

Cdt1 is a novel MT-binding protein. (A) Disorder prediction in human Cdt1. Disordered prediction (sum of 10 programs) is plotted as a function of residue number. (B) Seven homology models of full-length Cdt1; colored from N terminus (blue) to C terminus (red) with regions of low confidence or low overlap shown in translucent. Conserved WHM and WHC domains shown and aligned in gray and black, respectively, for clarity. Magnified is the PyMol rendered cartoon representation showing superimposition of the C-terminal human Cdt1 (410–546 aa, in red) generated using Phyre2 server with available NMR structure (PDB ID: 2KLO; in green) of mouse C-terminal Cdt1 (420–557 aa). (C) Constructs used in this study, full-length Cdt1 (1–546 aa), its deletion variant devoid of N-terminal 92 aa (92–546), and N- and C-terminal deletion variants generated in fusion with an N-terminal GFP tag for expression in bacteria are shown as black bars. Disordered domain shown in blue, WHM domain shown in yellow, and WHC domain shown in red. (D) SDS-PAGE (15%) of the indicated purified recombinant proteins along with native tubulin purified from porcine brain. M, molecular mass standard; *, degradation bands (confirmed by Western blot) in Cdt1^92–410. Arrows point toward the band corresponding to the protein of interest. (E) Western blots showing MT cosedimentation for Cdt1^92–546 (GFP-tagged) and full-length Cdt1^1–546 (without any tag), 1 µM each; purified from bacteria with the indicated concentrations of taxol-stabilized MTs (in μM). Samples fractionated as supernatant (S) and pellet (P) were analyzed by Western blot and probed with antibodies against 6×-His tag or anti-Cdt1 as indicated in each case to detect Cdt1. Ponceau S–stained tubulin. (F) Quantification (mean ± SD, n = 3) of the Cdt1^92–546 and Cdt1^1–546 blots using ImageJ. The 95% CI values obtained for the fit were 0.60–0.94 for B_max, 0.37–4.04 for K_d, and 0.07–0.29 for the background for the former construct; and 0.63–0.79 for B_max, 1.33–3.25 for K_d, and 0.08–0.18 for the background for the latter construct. (G) Representative Western blots of the binding of indicated deletion fragments to MTs in a cosedimentation assay with the indicated MT concentrations (top). (H) Quantification of binding (mean ± SD, n = 3) of the deletion fragments to MTs. (I) Quantification of MT-binding of Cdt1’s smallest C-terminal fragment, Cdt1^410-546, and a representative Western blot (top) from three independent experiments (mean ± SD, n = 3). (J) Blot overlay assay to study Cdt1–Hec1 interaction. Indicated proteins (0.5 µg each) were loaded as bait on 18% SDS-PAGE gel, transferred to nitrocellulose membrane, and blocked with 5% SM-TBST. Hec1::Nuf2-His dimeric complex (1 µg; a generous gift from A. Desai and D. Cheerambathur, University of California, San Diego, La Jolla, CA) was overlaid as prey protein on the membrane for 12 h at 4°C. The blot was washed and probed with anti-Hec1 antibody (1:2,000; 9G3; Abcam) followed by chemiluminescence. The same blot was probed with Ponceau S.