Mutations affecting beta-tubulin folding and degradation.
Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-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 beta-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 beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-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 beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.
Wang
Yaqing
Y
Department of Integrative Biology and Pharmacology, The University of Texas Medical School, Houston, Texas 77030.
Tian
Guoling
G
Department of Biochemistry, New York University Medical Center, New York, New York 10016.
Cowan
Nicholas J
NJ
Department of Biochemistry, New York University Medical Center, New York, New York 10016.
Cabral
Fernando
F
Department of Integrative Biology and Pharmacology, The University of Texas Medical School, Houston, Texas 77030. Electronic address: fcabral@uth.tmc.edu.
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D007976:Leupeptins; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex; D017510:Protein Folding; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007976:Leupeptins; D014404:Tubulin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde