In vitro analysis of microtubule assembly of isotypically pure tubulin dimers. Intrinsic differences in the assembly properties of αβII, αβIII, and αβIV tubulin dimers in the absence of microtubule-associated proteins

Microtubule assembly of different β tubulin isotypes in the presence of 4 M glycerol and 6 mM magnesium ion demonstrates significantly different characteristics. αβII and αβIV assembled faster and to a greater extent than did unfractionated phosphocellulose-purified tubulin (PC-tubulin). Microtubule assembly from αβIII showed a distinctive delay in nucleation, proceeded at a slower rate than those of the other β tubulin isotypes, and had the highest critical concentration. However, treatment of β tubulin isotypes with subtilisin to remove the C-terminal domain of the tubulin dimer abolished these differences in microtubule assembly pattern and enhanced self-assembly. The kinetic analysis of microtubule elongation of different β tubulin isotypes also showed significant differences. Elongation of αβIII from microtubule seeds had a lower apparent K(a) and a lower apparent K(d) than did αβII and αβIV. The dynamic behaviors of different β tubulin isotypes were qualitatively similar to each other and fit the dynamic instability model. However, microtubules formed from αβIII appeared to be less dynamic than microtubules formed from other β tubulin isotypes. Our results suggest that the βIII isotype might have a different conformation than do the other β tubulin isotypes. The distinctive nucleation and elongation behaviors of the αβIII dimers demonstrated in vitro may have a significant influence on microtubule functions in vivo.