Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex

Summary Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.
Highlights • Misato interacts biochemically with the Tubulin Chaperone Protein-1 (TCP-1) complex • Misato stabilizes the TCP-1 complex, possibly by filling its Tubulin-folding cavity • Loss of Misato or TCP-1 complex subunits leads to similar mitotic phenotypes • In the absence of Misato, Tubulin is unstable and unable to efficiently polymerize
Palumbo et al. show the conserved protein Misato interacts with the Tubulin Chaperone Protein-1 (TCP-1) complex in Drosophila. In the absence of Misato, TCP-1 subunit levels are reduced, functional Tubulin is unstable, and mitotic microtubule generation is compromised. The predicted structure of Misato suggests it acts as a molecular placeholder in the absence of Tubulin, stabilizing the TCP-1 complex.