Newly-synthesized beta-tubulin demonstrates domain-specific interactions with the cytosolic chaperonin

Abstract

Tubulin folding requires two chaperone systems, i.e., the 900 kDa cytosolic chaperonin referred to as the TCP-1 complex or TRiC which facilitates folding of the alpha- and beta-tubulin subunits and a ca. 180 kDa complex which facilitates further assembly into heterodimer. beta-Tubulin mutants were expressed in rabbit reticulocyte lysates, and the effect of C-terminal, N-terminal, and internal deletions on the binding of beta-tubulin polypeptides to the 900 and 180 kDa complexes was ascertained. Proteolytic studies of chaperonin-bound beta-tubulin were also implemented. These studies support the concept of quasi-native chaperonin-bound intermediates [Tian et al. J. Biol. Chem. (1995) 270, 1-4]. Three "domains" similar in size to the domains in the native protein were implicated in facilitated folding: i.e., an internal or "M-domain" composed of residues approximately 140-260 which binds to TRiC; a "C-domain" composed of residues approximately 300-445 which interacts less strongly with TRiC and may contain regulatory sequences for tubulin release from the chaperonin; and an "N-domain" composed of residues approximately 1-140 which apparently does not interact with TRiC but does interact with the 180 kDa complex. The major TRiC-interacting region, residues approximately 150-350 (the "interactive core"), overlapped portions of the M- and C-domains and included a putative hydrophobic-rich interdomain segment which may be a preferential site of interaction with TRiC. This segment may also be important for microtubule assembly and/or tubulin dimer formation. Removal of two residues from the N-terminal end or ca. 27 residues from the C-terminal and caused the polypeptide to arrest on TRiC. It is proposed that N- and C-terminal regions of beta-tubulin structurally interact with TRiC-binding region approximately 150-350 to inhibit binding to TRiC.