Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle

The hetero-octameric protein complex, Augmin, recruits gamma-Tubulin ring complex (gamma-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of in vivo Augmin from Drosophila and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and gamma-TuRC. We tested these predictions biochemically and in the Drosophila embryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the gamma-TuRC protein, Dgp71WD, and are required for the accumulation of gamma-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation.