1. In vitro phosphorylation of condensin SMC requires the ATPase domain.
In vitro phosphorylation of condensin SMC requires the ATPase domain. (a) Illustration of condensin SMC heterodimer (i) and hetero-pentameric holocondensin, consisting of an SMC dimer and a non-SMC trimer (ii). The ATPase domain containing the Walker motif is located in the head domain. The head domain interacts with non-SMC subunits. (b) The time course of γP32 labelling of Cut3–Cut14, Cut3 and Cut14 subunits in the presence of γP32-ATP. See text. (c) To confirm that γP32-labelled protein was actually phosphorylated, phage λ phosphatase (PPase) was employed to treat proteins after the reaction with γP32-ATP. I and NaF are phosphatase inhibitors. (d) A large excess of ATP and other nucleotides were added. See text. (e) Four Cut3–Cut14 mutant proteins overproduced in S. pombe were purified as a complex by affinity chromatography and incubated with radiolabelled γP32-ATP (i). The double mutant protein Cut3 K161I–Cut14 K38T-was hardly phosphorylated. CBB-stained protein bands are seen at the bottom (ii). (f) Single Cut3 K161I (i) and Cut14 K38T (ii) mutant protein were not γP32 phosphorylated, whereas wild-type, single subunits Cut3 and Cut14 were heavily phosphorylated.
Yuko Akai et al. Open Biol. 2014;4:140193