Figure 1.

The C-terminal domain and neck of MCAK are necessary for efficient MT depolymerization activity. (A) Schematic diagram of expressed and purified MCAK proteins. The GFP tag is located on the N-terminus but was omitted from the diagrams for simplicity. (B and C) GMPCPP stabilized MTs (1 μM) were incubated with increasing concentrations of enzyme (0–1 μM) for 30 min in saturating MgATP at room temperature. MTs were sedimented, and the soluble tubulin heterodimer removed. Equal volumes of soluble tubulin and MT pellets were electrophoresed on 10% SDS polyacrylamide gels, and the gels were stained with Colloidal Coomassie Blue. The percentage of soluble tubulin was quantified from the stained gels and plotted against the log of the enzyme concentration. The four-parameter logistic equation was used to determine the EC₅₀ concentrations of each enzyme from four or more individual experiments. The curves represent the best fit curve to the data with each point represented by the mean ± SEM.