Tubulin-Specific Chaperones

The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.