Mutating the interprotofilament interface allows microtubules to assemble in GDP

Microtubule dynamic instability, driven by GTP turnover, allows microtubules in cells to reorganise themselves adaptively. GTP promotes the assembly of tubulin into microtubules, but exactly how it does so is controversial. In some models, GTP mainly supports the assembly of tubulin into single protofilaments, as in prokaryotic tubulins. In others, GTP mainly supports the formation of lateral bonds between protofilaments. To investigate, we mutated the interprotofilament interface in human α1bβ3 and α1bβ4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the α1bβ3 M-loop or its binding pocket into α1bβ4b tubulin creates tubulins that assemble in 1 mM GDP. We accordingly propose that GTP- and GDP-tubulins are captured equivalently at the tips of microtubules, but then differentially retained, based on their differing abilities to form stable interprotofilament bonds. This biased retention mechanism allows mosaic lattices to be built and dynamic instability to be tuned.