Tubulin Cofactors Form a Multi-Protein Platforms that Regulate the Soluble Tubulin Pool and Promote Microtubule Polymerization

Abstract

The Microtubule (MT) cytoskeleton mediates intracellular organization and trafficking, and is responsible for force generation during cell division and migration in eukaryotes. The building block for MT polymerization is the as-tubulin heterodimer, which is present as a tightly regulated soluble pool in the cytoplasm. Despite the importance of as-tubulin heterodimer in the regulation of MT dynamics, it remains unclear how nascent and folded a and s-tubulin are assembled and activated into a single heterodimer configuration that universally dictates dynamic MT polymerization. It remains unknown how five conserved tubulin cofactors (TBC-A,B,C,D and E) and a dedicated Arl2 G-protein promote as-tubulin biogenesis, activation and degradation, and how such activities impact MT function. In contrast to a long-standing hypothesis in which individual tubulin cofactors bind sequentially to a and s-tubulin monomers and assemble as-tubulin dimers through dynamic interactions, we show based on biochemical and structural studies that multiple tubulin cofactors and Arl2 form multi-subunit platforms for as-tubulin dimer assembly, activation and degradation. We show that multi-subunit tubulin cofactor and Arl2 platform are soluble as-tubulin regulators that are powered by GTP hydrolysis cycles. We have determined tubulin cofactor platform structure, conformational changes upon tubulin dimer binding, and the mechanism of GTP hydrolysis activation. Surprisingly, we further show, using reconstitution of these complexes with soluble tubulin dimer and dynamic MTs, that they enhance as-tubulin polymerizing state at MT plus ends in a manner dependent on Arl2 and tubulin GTP hydrolysis. Our data surprisingly suggest tubulin cofactors are potent regulators of soluble tubulin dimer state, and promote soluble tubulin activation required for all MT dynamics. Our model explains long-standing cell biology and genetics data about the roles of tubulin cofactors in regulating the soluble as-tubulin pool and MT homeostasis.