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Fig. 5. E-like disrupts the tubulin heterodimer in vitro. (A) E-like mediated disruption of the tubulin heterodimer in vitro. Purified E-like (El) and cofactor E (E) were incubated with depolymerized bovine brain tubulin (tub). The reaction products were resolved by non-denaturing (N.D.) gel electrophoresis (upper panel) and SDS-PAGE (lower panel). Note that the absence of a band corresponding to purified cofactor E on the native gel is a result of the migration of this protein towards the cathode under these conditions. 0.1x, 0.3x, 1x, 2x and 5x refer to relative molar concentrations of either E-like or cofactor E compared with tubulin. (B) E-like induces aggregation of tubulin in vitro. [³⁵S]tubulin was incubated with the indicated amounts of E-like (El) and the reaction products were analyzed under non-denaturing conditions. (C) E-like prevents MT assembly in vitro. Micrographs (magnification 1300x) show MTs (arrowed) assembled from axoneme fragments 5-10 minutes after warming samples to 37°C. Pre-incubation with 2 µM E-like significantly reduced the assembly of 11 µM tubulin. MTs did not assemble after preincubation with higher concentrations of E-like. The histogram shows the number of MTs assembled per axoneme end for 11 µM tubulin preincubated for 1 hour with the indicated concentrations of E-like. Data shown are the mean±s.d. from 100 axoneme ends per condition.

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