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. 2021 Jul 22;40(18):e107516. doi: 10.15252/embj.2020107516

Figure 1. Plx1 binds to Apc1‐loop500 via its polo‐box domain.

Figure 1

  1. Schematic diagram of Apc1 is shown. Two loop domains (loop300, 294–399; loop500, 515–584) and PC repeat domain are shown in yellow and in cyan, respectively. Multiple alignment of sequences of Apc1‐loop500 containing PBMs (Orange) and B56‐binding site (green) is shown. Introduced alanine substitutions are indicated as A in red. Hs, Homo sapiens human; Pt, Pan troglodytes chimpanzee; Mm, Mus musculus mouse; Gg, Gallus gallus chicken; and Xt, Xenopus tropicalis frog. Conserved or similar amino acids are shown with an asterisk (*) or dot (.), respectively.
  2. Binding assay using MBP‐fused Apc1‐loop500 fragments. MBP‐fused Apc1‐loop500 WT or its derivatives (T532A, T539A, 2A (T532A/T539A) and S558A) was incubated with anaphase extracts supplemented with non‐degradable cyclin B at 23°C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS–PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining.
  3. Quantification of (B). The bar graph is quantification of bound Plx1. The intensities of Apc1‐loop500 WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.
  4. Specific binding of Plx1‐PBD to Apc1‐loop500. MBP‐fused Apc1‐loop500 WT or its derivatives 2A (T532A/T539A) was incubated in anaphase extracts supplemented with non‐degradable cyclin B at 23°C for 1 h and further incubated in the presence of 10 µg of WT Plx1‐PBD for 15 min. The bound proteins were recovered by amylose beads, separated by SDS–PAGE and detected by Coomassie brilliant blue staining (CBB). Purified proteins (Plx1‐PBD and Apc1‐loop500) were run as a standard.
  5. Quantification of (D). The bar graph is quantification of bound Plx1‐PBD. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.
  6. Plx1‐PBD, but not its Pincer mutant, binds to Apc1‐loop500. MBP‐fused Apc1‐loop500 WT was incubated in anaphase extracts supplemented with non‐degradable cyclin B at 23°C for 1 h and further incubated in the presence of indicated amounts of WT Plx1‐PBD or Plx1‐PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads and analysed as described in (D). The bound Plx1‐PBD is shown as stoichiometries (the bound Plx1‐PBD per Apc1‐loop500) determined from Fig EV1.
  7. CDK‐dependent binding of Plx1‐PBD to the Apc1‐loop500. MBP‐fused Apc1‐loop500 was incubated in the presence or absence of purified Cdk2/cyclin A at 30°C for 2 h. MBP‐fused Apc1‐loop500 (CDK+/−) was isolated by amylose beads and further incubated with indicated amounts of WT Plx1‐PBD on ice for 10 min. The bound proteins were recovered by amylose beads and analysed as described in (D).