Newly-Synthesized β-Tubulin Demonstrates Domain-Specific Interactions with the Cytosolic Chaperonin

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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 α- and β-tubulin subunits and a ca. 180 kDa complex which facilitates further assembly into heterodimer. β-Tubulin mutants were expressed in rabbit reticulocyte lysates, and the effect of C-terminal, N-terminal, and internal deletions on the binding of β-tubulin polypeptides to the 900 and 180 kDa complexes was ascertained. Proteolytic studies of chaperonin-bound β-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 ∼140−260 which binds to TRiC; a “C-domain” composed of residues ∼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 ∼1−140 which apparently does not interact with TRiC but does interact with the 180 kDa complex. The major TRiC-interacting region, residues ∼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 end caused the polypeptide to arrest on TRiC. It is proposed that N- and C-terminal regions of β-tubulin structurally interact with TRiC-binding region ∼150−350 to inhibit binding to TRiC.