Roles of beta-tubulin residues Ala428 and Thr429 in microtubule formation in vivo

Abstract

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).

FIGURE 1.

Sequence alignment of human βIa and βIII tubulin isotypes. Human βIa and βIII tubulin isotypes were aligned with ClustalX 1.83 and processed with BioEdit. Hyphens denote identical residues between sequences.

FIGURE 2.

Microtubule incorporation of βIII tubulin is abrogated with C-terminal tail truncations larger than 22-amino acids. Panel a, HeLa cells were transfected with βIII wild type (A), βIII CΔ5 (B), βIII CΔ10 (C), βIII CΔ20 (D), βIII CΔ21 (E), βIII CΔ22 (F), βIII CΔ23 (G), βIII CΔ24 (H), βIII CΔ25 (I), and βIII CΔ30 (J). The positive control was expressing V5-His tagged β-galactosidase (K), whereas the negative control was of nontransfected HeLa cells (L). Twenty-four hours post-transfection, the cells were fixed and labeled with an antibody to V5 tag followed by Cy3-conjugated goat anti-mouse and visualized at 60× magnification. Microtubules are shown in red, and nuclei are stained blue. Insets, visualization of cortical microtubules after 2× digital expansion. Panel b, inflection point of microtubule and non-microtubule-forming cells (βIII CΔ22 (A) and βIII CΔ23 (B), respectively) from transfected HeLa culture. Bar, 10 μm.

FIGURE 3.

Microtubule incorporation of βI tubulin is abrogated with C-terminal tail truncations larger than 16 amino acids. Human βI wild type (A), βI CΔ16 (B), and βI CΔ17 (C) were transfected in HeLa cells. Twenty-four hours post-transfection, the cells were fixed and labeled with an antibody to V5 tag followed by Cy3-conjugated goat anti-mouse and visualized at 60× magnification. Insets, visualization of cortical microtubules after 2× digital expansion. Notice that none of the cells transfected with βI CΔ17 (C) were able to form microtubules. Bar, 10 μm.

FIGURE 4.

Chimeras of βI and βIII tubulin do not require the opposing, full-length, C-terminal tail for microtubule incorporation. Chimeras of human βI and βIII tubulin were constructed and transfected to show that beyond Ala⁴²⁸ (N-QQYQDA⁴²⁸), the C-terminal tails are interchangeable and do not abolish microtubule formation. Immunofluorescence of HeLa cells transfected with βIII+CβI FL (A), βI+CβIII FL (B), βI+CβIII CΔ5 (C), βI+CβIII CΔ9 (D), and βI+CβIII CΔ13 (E) indicates that all chimeric constructs enter microtubules. A chimera, βI+CβIII FL (F), was created lacking the V5 tag to show that the tag does not interfere with microtubule formation. The cells in A–E were stained with antibody to the V5 tag; the cells in F were stained with the antibody SDL.3D10 to βIII. Twenty-four hours post-transfection, the cells were fixed and labeled with an antibody to V5 tag followed by Cy3-conjugated goat anti-mouse and visualized at 60× magnification. Insets, visualization of cortical microtubules after 2× digital expansion. Microtubules are shown in red, and nuclei are shown in blue. Notice that the full-length, chimeric tail is not required for microtubule formation (C–E). Bar, 10 μm.

FIGURE 5.

Quantitative analysis of HeLa cells transfected with βI, βIII, and chimeric β-tubulin constructs were calculated with respect to microtubule-forming versus non-microtubule-forming. Immunofluorescence of transiently transfected HeLa cells were visualized at 60× magnification and quantitated based on the ability of the ability of each transfected construct to enter microtubules. βIII tubulin truncations (A), βI tubulin truncations (B), and chimeras (C) were counted and expressed as percentages of microtubule-forming versus non-microtubule-forming cells. For each construct, four random fields were averaged with standard deviation. Notice that βIII CΔ23 and βI CΔ17 (A and B, respectively) do not form any microtubules. In addition, all chimeras, including truncated chimeras βI+CβIII CΔ5, βI+CβIII CΔ9, and βI+CβIII CΔ13, formed microtubules robustly (C). Empty vector did not form microtubules (C).

FIGURE 6.

Co-immunoprecipitation and Western analysis of βI, βIII, and chimeric β-tubulin constructs. HeLa cells were transiently transfected with wild type or the indicated C-terminally truncated βIII tubulin (A), βI tubulin (B), or chimeric β-tubulin (C). Expression of βI, βIII, and chimeric β-tubulin in HeLa cells were analyzed by Western blotting with antibody to V5 (A–C, top panel) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control (A–C, middle panel). The samples were immunoprecipitated and analyzed by Western blotting with antibody to β-tubulin (A–C, bottom panels). C, *, βI+CβIII FL construct that does not contain C-terminal V5-His tag. **, empty vector expressing V5-His-tagged β-galactosidase. Notice that βI CΔ17, βIII CΔ23, βIII CΔ24, βIII CΔ25, and βIII CΔ30 were all able to precipitate α-tubulin. IB, immunoblot.

FIGURE 7.

Location of Asp⁴²⁷ at the C terminus of β-tubulin. This representation of the αβ-tubulin dimer, taken from the structure by Lowe et al. (5) (Protein Data Bank code 1JFF), indicates the location of Asp⁴²⁷ near the end of helix H12. The Asp⁴²⁷ side chain of β-tubulin is shown in blue. This structure was generated using PyMOL (72).