Tubulin-specific chaperones: components of a molecular machine that assembles the ?/? heterodimer.

ABSTRACT: The tubulin heterodimer consists of one ?- and one ?-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 ?- and ?-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of ?-tubulin between TBCA and TBCD, and of ?-tubulin between TBCB and TBCE, resulting in the formation of TBCD/? and TBCE/?, respectively. The latter two complexes interact, forming a supercomplex (TBCE/?/TBCD/?). Discharge of the native ?/? heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound ?-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 ?- and ?-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate ?- or ?-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.

SUBMITTER: Tian G 

PROVIDER: S-EPMC4961357 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature