Tubulin cofactors and Arl2 form a cage-like chaperone that catalyzes αβ-tubulin assembly through GTP hydrolysis: Reconstitution of new molecular machine

The building block of microtubules is composed of two proteins called α-tubulin and ß-tubulin which are assembled into a single polarized αß-tubulin subunit. Microtubules are very dynamic structures that can rapidly change length as individual αβ-tubulin units are either added or removed to their growing or shrinking ends. The assembly of aß-tubulin has for long remained a mystery despite extensive genetic and cell biology studies. Several proteins known as tubulin cofactors and an enzyme called Arl2 help to build a vast pool of αβ-tubulin that are able attach to the microtubules. It has remained unknown how this collection of proteins drives αβ-tubulin biogenesis and degradation inside cells. And the roles of the conserved tubulin cofactors and Arl2 remain unknown.

Our group used a combination of biochemical reconstitution of all tubulin cofactors and Arl2 GTPase in combination with negative stain electron microscopy and biochemical techniques to study how the tubulin cofactors and Arl2 are organized, and their role in the assembly of aß-tubulin and polymerization of dynamic microtubules inside living yeast cells. Our biochemical reconstitution has revealed that Arl2 and two tubulin cofactors TBCD and TBCE associate with each other to form a stable ‘complex’ that has a cage-like structure. A molecule of αβ-tubulin binds to the complex, followed by another cofactor called TBCC. The binding of αβ-tubulin and TBCC activates the enzyme activity of Arl2, which releases the energy needed to alter the shape of the αβ-tubulin in a larger ternary complex. We also found that inactivating Arl2 GTP hydrolysis inhibits the dissociation of this ternary complex and introducing this mutant form of Arl2 that lacked enzyme activity inside yeast cells had problems forming microtubules characterized by poor polymerization due to pausing and defects in cell division.

Together, our findings show that the tubulin cofactors and Arl2 form a complex that regulates the assembly and maintenance of αβ-tubulin. The next challenges are : 1) to understand how this regulation occurs by determining the high resolution structures of these assemblies and  2) to determine how this regulation influences the way that microtubules grow and shrink inside cells.

Published in eLife.