Assembly of microtubules from nucleotide-depleted tubulin

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Abstract
In vitro assembly of microtubules from tubulin is considered to have an absolute requirement for added GTP (or a non-hydrolysable GTP-analogue)1 involving binding at the E(exchangeable)-site located on the β-subunit of the tubulin dimer2. By contrast, GDP inhibits assembly. Nucleotide hydrolysis has been implicated in the dynamic properties of microtubules3,4, treadmilling5 and mechanical coupling6. Here we demonstrate that assembly is not necessarily dependent on the presence of GTP at the E-site; microtubules can be formed efficiently in the absence of GTP in the presence of pyrophosphate. These microtubules, which have normal morphology and lability at cold temperatures, contain N(non-exchangeable)-site GTP and a significant proportion of E-site GDP. This demonstrates the possibility of direct incorporation of GDP-containing tubulin dimer during assembly which probably derives from microtubule-associated protein (MAP)-containing oligomers. This finding has important implications for the mechanism of microtubule elongation. The effects of pyrophosphate suggest that charge neutralization by the bidentate ligand is an essential step in promoting microtubule assembly, and that this interaction involves only a minimal conformational change in the protein.