Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Abstract

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo-electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.

FIGURE 1:

Tubulin acetylation does not affect the amount of polymerization or gross morphology of microtubules. Representative cryo-EM images of (A) acetylated and (B) deacetylated dynamic microtubules. Scale bar, 50 nm. (C) Protofilament number distribution of microtubules polymerized after acetylation or deacetylation into dynamic or Taxol-stabilized microtubules. Arrow shows largest observed change.

FIGURE 2:

Acetylation state does not alter the structure of dynamic microtubules. (A) End-on view of the deacetylated, dynamic microtubule reconstruction with the docked electron crystallographic structure of tubulin (1JFF). (B) Inside view (from the microtubule lumen) of the difference maps between acetylated and deacetylated reconstructions contoured at 3σ, with extra density in acetylated microtubules shown in blue and extra density in deacetylated microtubules shown in red. The differences are superimposed on the cryo-EM reconstruction of deacetylated microtubules, shown as gray isosurface. The location of the loop containing the modified residue is indicated by the circle. (C) Outside view, colors as in B. α-Tubulin is shown in green and β-tubulin in blue.

FIGURE 3:

Preventing averaging of α- and β-tubulin by kinesin decoration does not reveal any difference between acetylation states. (A) End-on view of the deacetylated, dynamic microtubule reconstruction with kinesin. (B) Inside and (C) outside views of an isolated tubulin dimer, with differences superimposed as in Figure 2. No significant differences were observed within the tubulin dimer. The location of the loop containing the modified residue is indicated by the circle. α-Tubulin is shown in green, β-tubulin in blue, and kinesin (1BG2) in purple.

FIGURE 4:

αTAT1 accessibility to its binding sites on tubulin polymers. (A) SDS gel (top) and quantitation (bottom) of pelleting assays showing that αTAT1 binds similarly to tubulin polymers assembled with vinblastine (coils) or Taxol (microtubules). (B) Shearing microtubules to create more microtubule ends does not increase binding of αTAT1 to microtubules. Data from three independent cosedimentation assays. Error bars, SD.

FIGURE 5:

Effect of αTAT1 binding on microtubule assembly and bundling. (A) Cryo-EM image showing that the presence of αTAT1 during the assembly of dynamic microtubules gives rise to large protein structures around the microtubule lattice (black arrow) and promotes bundling (white arrow). (B) Negative-stain EM image of microtubules copolymerized with αTAT1 in the presence of Taxol at a 1:5 αTAT1:tubulin molar ratio, showing incomplete microtubule closure and numerous defects. Scale bar, 50 nm.

FIGURE 6:

αTAT1 binding to microtubules is affected by the absence of the C-terminal tails of tubulin. SDS gel (top) and quantitation (bottom) of pelleting assays showing that αTAT1 cosediments less with subtilisin-treated microtubules than with untreated tubulin. Data from three independent cosedimentation assays. Error bars, SD.

FIGURE 7:

αTAT1 binding to microtubules is not affected by the acetylation state of tubulin. αTAT1(D157N) cosedimented similarly with microtubules polymerized from tubulin that was previously acetylated or deacetylated. Ac, acetylated; dAc, deacetylated; Un, untreated.