Roles of beta-tubulin residues Ala428 and Thr429 in microtubule formation in vivo.
The C termini of beta-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human betaI and betaIII tubulin. Transient transfection of HeLa cells with the mutants shows that mutants with deletions of up to 22 residues from betaIII and 16 from betaI can assemble normally. Interestingly, removal of the next residue (Ala(428)) results in a complete loss of microtubule formation without affecting dimer formation. C-terminal tail switching of human betaI and betaIII tubulin suggests that C-terminal tails are functionally equivalent. In short, residues outside of 1-429 of human beta-tubulins make no contribution to microtubule assembly. Ala(428), in the C-terminal sequence motif N-QQYQDA(428), lies at the end of helix H12 of beta-tubulin. We hypothesize that this residue is important for maintaining helix H12 structure. Deletion of Ala(428) may lead to unwinding of helix H12, resulting in tubulin dimers incapable of assembly. Thr(429) plays a more complex role. In the betaI isotype of tubulin, Thr(429) is not at all necessary for assembly; however, in the betaIII isotype, its presence strongly favors assembly. This result is consistent with a likely more complex function of betaIII as well as with the observation that evolutionary conservation is total for Ala(428) and frequent for Thr(429).
Joe
Patrick A
PA
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Banerjee
Asok
A
Ludueña
Richard F
RF
D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D020033:Protein Isoforms; C498585:TUBB3 protein, human; D014404:Tubulin