Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers.
Cell-free translation of beta-tubulin mRNA generates full length beta-tubulin polypeptides distributed in three molecular forms: a high molecular weight lysate-associated form, the free beta-tubulin subunit, and the alpha beta-heterodimer (Yaffe, M.B., Farr, G. W., and Sternlicht, H. (1988) J. Biol. Chem. 263, 16023-16031). A quantitative assay system for these three forms was developed and used to measure the rates of incorporation/exchange of the newly synthesized free beta-subunit and the high molecular weight form into tubulin heterodimers following incubation of the 35S-translation products with unlabeled bovine tubulin dimer. This exchange process was found to be slow and strongly temperature-dependent. The half-lives for exchange ranged from 12.5 min at 37 degrees C to 17.5 h at 0 degree C with a measured activation energy of 22.5 kcal/mol. Microtubule-associated proteins appeared to play no role in the exchange process, since identical exchange rates were observed regardless of whether microtubule protein or phosphocellulose-purified tubulin was used as the source of tubulin dimer. Surprisingly, the exchange rates were found to be independent of dimer concentration. We interpret these results as evidence for a rate-limiting, slow conformational change that occurs within the newly synthesized beta-subunits prior to their association with alpha-tubulin to generate the alpha beta-hetero-dimer.
Yaffe
M B
MB
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.
Farr
G W
GW
Sternlicht
H
H
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D007700:Kinetics; D046911:Macromolecular Substances; D008962:Models, Theoretical; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013816:Thermodynamics; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism
D046911:Macromolecular Substances; D012333:RNA, Messenger; D014404:Tubulin