Mutating the interprotofilament interface allows microtubules to assemble in GDP

Abstract

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 1b{beta}3 and 1b{beta}4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the 1b{beta}3 M-loop or its binding pocket into 1b{beta}4b 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. One sentence summaryMicrotubules grow not by biased capture of GTP tubulins, but by biased retention of tubulins that form stable interprotofilament bonds.