Fig. 2. Functional comparison between E-like and cofactor E. (A) E-like cannot substitute for cofactor E in tubulin folding reactions. ³⁵S-labeled, unfolded -tubulin (left panel) or ß-tubulin (right panel) probes generated by expression in E. coli were presented to CCT by sudden dilution from denaturant in the presence of ATP and GTP plus the indicated cofactors (B,C,D) and either cofactor E (E) or increasing concentrations of E-like (El). 1x, 2x and 4x denote the fold molar excess of E-like compared with cofactor E. (B) E-like cannot substitute for cofactor E in tubulin refolding reactions. Tubulin refolding reactions containing various tubulin-specific chaperones were carried out in the presence of [³²P]GTP as described (Bartolini et al., 2002). In A and B, reaction products were resolved by non-denaturing gel electrophoresis. Migration positions of various tubulin-containing species are shown. Note the absence of labeled native tubulin heterodimers (tub) in lanes in which E-like is substituted for E. Dß, cofactor D/ß-tubulin; B, cofactor B/-tubulin. (C) E-like does not stimulate the tubulin-GAP activity of cofactors C and D. The tubulin-GAP activity of cofactors C and D was measured as described (Tian et al., 1999) either alone (open circles), in the presence of cofactor E (filled diamonds) or in the presence of E-like (crosses).