Mutations affecting β-tubulin folding and degradation

Abstract

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) β-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable β-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective β-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with β-tubulin in purified αβ heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded β-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of β-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.