FIGURE 2.

pCopN inhibits microtubule assembly by sequestering tubulin and by delaying nucleation. A, the pCopN constructs used in this study. The effect of mutations was studied in the framework of the D29 construct. B, critical concentration plots for microtubule assembly in the absence (●) and in the presence of 5 μm FL_pCopN (■) or D84 (▴) (left) or in the presence of 3 μm (▴) or 5 μm (■) D29 (right). C, time course of tubulin assembly in the presence of increasing amounts of tubulin-FL_pCopN complex. Left, the assembly of 10 μm tubulin or of tubulin/pCopN mixtures at the indicated concentrations was assayed turbidimetrically. The temperature was switched from 4 to 37 °C after 1 min of recording time in each case. Right, the same experiments but with 15 μm tubulin (solid line) or with mixtures of tubulin/pCopN at 20 μm/5 μm (dotted line) or 30 μm/15 μm (dashed line) concentrations. Arrowheads, time of the reverse temperature switch (same time for the 15:0 and the 20:5 tubulin/pCopN samples). The 1:1 tubulin/pCopN stoichiometry ensured that “free” tubulin was at the same concentration at the beginning of each set of experiments (either 10 μm (left) or 15 μm (right)). D, dual mechanism of pCopN on microtubule assembly. pCopN inhibits microtubule elongation by sequestering tubulin (B). The inhibition of microtubule nucleation is not solely explained by tubulin sequestration because increasing the tubulin-pCopN complex concentration for a given free tubulin concentration delays assembly (C). This implies that pCopN or its complex with tubulin interacts with the nucleus to yield a non-productive (as schematized here) or less productive nucleus for microtubule assembly ([Nuc]*). AU, absorbance units.