Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15–20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth is 80–85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella $\text{1}\text{3}\text{–}\text{1}\text{2}$ normal length. Nevertheless, in vivo labeling with ¹⁴C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction of tubulin synthesis.Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.     

Tubulin Subunits Exist in an Activated Conformational State Generated and Maintained by Protein Cofactors

The production of native α/β tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on β-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from α-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of α-tubulin folding in vitro. This enabled an integrated study of α- and β-tubulin folding: we find that the pathways leading to the formation of native α- and β-tubulin converge in that the folding of the α subunit requires the participation of cofactor complexes containing the β subunit and vice versa. We also show that sequestration of native α-or β-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining α-and β-tubulin polypeptides in an activated conformational state required for the formation of native α/β heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.     

Formation and Function of the Rbl2p–β-Tubulin Complex

The yeast protein Rbl2p suppresses the deleterious effects of excess β-tubulin as efficiently as does α-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with β-tubulin that does not contain α-tubulin, thus defining a second pool of β-tubulin in the cell. Formation of the complex depends upon the conformation of β-tubulin. Newly synthesized β-tubulin can bind to Rbl2p before it binds to α-tubulin. Rbl2p can also bind β-tubulin from the α/β-tubulin heterodimer, apparently by competing with α-tubulin. The Rbl2p–β-tubulin complex has a half-life of ~2.5 h and is less stable than the α/β-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing β-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p–β-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.     

Evidence for new C-terminally truncated variants of α- and β-tubulins

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the –EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same –EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with –EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.     

Structural basis of tubulin tyrosination by tubulin tyrosine ligase

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL–tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton.     

Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin

Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides'' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.     

Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function

Mutations in the genes that encode α- and β-tubulin underlie many neurological diseases, most notably malformations in cortical development. In addition to revealing the molecular basis for disease etiology, studying such mutations can provide insight into microtubule function and the role of the large family of microtubule effectors. In this study, we use budding yeast to model one such mutation—Gly436Arg in α-tubulin, which is causative of malformations in cortical development—in order to understand how it impacts microtubule function in a simple eukaryotic system. Using a combination of in vitro and in vivo methodologies, including live cell imaging and electron tomography, we find that the mutant tubulin is incorporated into microtubules, causes a shift in α-tubulin isotype usage, and dramatically enhances dynein activity, which leads to spindle-positioning defects. We find that the basis for the latter phenotype is an impaired interaction between She1—a dynein inhibitor—and the mutant microtubules. In addition to revealing the natural balance of α-tubulin isotype utilization in cells, our results provide evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation and sheds light on a mechanism that may be causative of neurodevelopmental diseases.     

Centrosomes Isolated from Spisula solidissima Oocytes Contain Rings and an Unusual Stoichiometric Ratio of α/β Tubulin

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures ~25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase–specific phosphoepitopes (MPM-2), (b) α-, β-, and γ-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. γ-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little α- or β-tubulin copurified with centrosomes, and the ratio of α- to β-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain β-tubulin that is not dimerized with α-tubulin.     

Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas Flagella

A dramatic stimulation of synthesis of flagellar proteins occurs in Chlamydomonas following flagellar removal or experimentally induced resorption of the flagella into the cell. In this report we show that this stimulation involves an increase in the levels of mRNAs for tubulin and many other flagellar proteins. Total RNA and poly(A) RNA were isolated from cells after deflagellation or flagellar resorption, and were then translated in the reticulocyte lysate system. Two-dimensional gel analysis of the translation products demonstrates that the RNA-directed in vitro synthesis of α and β tubulins, and a number of other flagellar proteins, increases after deflagellation or flagellar resorption. Surprisingly, the α-tubulin synthesized in vitro does not co-migrate on two-dimensional gels with mature flagellar α-tubulin. Moreover, in vivo labeling experiments show that the major α-tubulin synthesized in the cell after deflagellation co-migrates with the major α-tubulin made in vitro, not with the major α-tubulin present in the flagella. These results suggest that flagellar α-tubulin is synthesized as a precursor, and undergoes post-translational modification before assembly into the flagella. In addition, we report that the synthesis of tubulin and other flagellar proteins can be specifically inhibited, as well as stimulated. Treatment of cells with IBMX, which induces flagellar resorption, causes a marked decrease in the levels of translatable mRNAs for tubulin and other flagellar proteins, without affecting levels of mRNAs for nonflagellar proteins.     

Tubulin isotypes optimize distinct spindle positioning mechanisms during yeast mitosis

Microtubules are dynamic cytoskeleton filaments that are essential for a wide range of cellular processes. They are polymerized from tubulin, a heterodimer of α- and β-subunits. Most eukaryotic organisms express multiple isotypes of α- and β-tubulin, yet their functional relevance in any organism remains largely obscure. The two α-tubulin isotypes in budding yeast, Tub1 and Tub3, are proposed to be functionally interchangeable, yet their individual functions have not been rigorously interrogated. Here, we develop otherwise isogenic yeast strains expressing single tubulin isotypes at levels comparable to total tubulin in WT cells. Using genome-wide screening, we uncover unique interactions between the isotypes and the two major mitotic spindle positioning mechanisms. We further exploit these cells to demonstrate that Tub1 and Tub3 optimize spindle positioning by differentially recruiting key components of the Dyn1- and Kar9-dependent mechanisms, respectively. Our results provide novel mechanistic insights into how tubulin isotypes allow highly conserved microtubules to function in diverse cellular processes.     

Tubulin Acetyltransferase αTAT1 Destabilizes Microtubules Independently of Its Acetylation Activity

Acetylation of α-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently, αTAT1, a member of the Gcn5-related N-acetyltransferase superfamily, has been identified as an α-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of αTAT1 with the aim of understanding the consequences of αTAT1-mediated microtubule acetylation. We demonstrate that α-tubulin is the major target of αTAT1 but that αTAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin. We further show that in mammalian cells, αTAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in an αTAT1 mutant with no acetyltransferase activity, suggesting that interaction of αTAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that αTAT1 has cellular functions that extend beyond its classical enzymatic activity as an α-tubulin acetyltransferase.     

The Motility of Axonemal Dynein Is Regulated by the Tubulin Code

Microtubule diversity, arising from the utilization of different tubulin genes and from posttranslational modifications, regulates many cellular processes including cell division, neuronal differentiation and growth, and centriole assembly. In the case of cilia and flagella, multiple cell biological studies show that microtubule diversity is important for axonemal assembly and motility. However, it is not known whether microtubule diversity directly influences the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, nor whether the effects on motility are indirect, perhaps through regulatory pathways upstream of the motors, such as the central pair, radial spokes, or dynein regulatory complex. To test whether microtubule diversity can directly regulate the activity of axonemal dyneins, we asked whether in vitro acetylation or deacetylation of lysine 40 (K40), a major posttranslational modification of α-tubulin, or whether proteolytic cleavage of the C-terminal tail (CTT) of α- and β-tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, can influence the activity of outer arm axonemal dynein in motility assays using purified proteins. By quantifying the motility with displacement-weighted velocity analysis and mathematically modeling the results, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic ciliary beat regulation.     

Tubulin acetylation increases cytoskeletal stiffness to regulate mechanotransduction in striated muscle

Microtubules tune cytoskeletal stiffness, which affects cytoskeletal mechanics and mechanotransduction of striated muscle. While recent evidence suggests that microtubules enriched in detyrosinated α-tubulin regulate these processes in healthy muscle and increase them in disease, the possible contribution from several other α-tubulin modifications has not been investigated. Here, we used genetic and pharmacologic strategies in isolated cardiomyocytes and skeletal myofibers to increase the level of acetylated α-tubulin without altering the level of detyrosinated α-tubulin. We show that microtubules enriched in acetylated α-tubulin increase cytoskeletal stiffness and viscoelastic resistance. These changes slow rates of contraction and relaxation during unloaded contraction and increased activation of NADPH oxidase 2 (Nox2) by mechanotransduction. Together, these findings add to growing evidence that microtubules contribute to the mechanobiology of striated muscle in health and disease.     

Tubulin Isotypes in Rye Roots Are Altered during Cold Acclimation

The cold stability of cortical microtubules in root-tip cells of winter rye (Secale cereale L. cv Puma) is altered by growth temperature (GP Kerr, JV Carter [1990] Plant Physiol 93:77-82). One hypothesis for the basis of this alteration is that different tubulin isotypes are present at different growth temperatures, and that the cold stability of microtubules is affected by these isotypic differences. We have explored the first part of this hypothesis by comparing protein extracts from roots of seedlings grown for 2 days at 22°C (nonacclimated) or for an additional 2 or 4 days at 4°C (cold-acclimated). Immunoblots of two-dimensional polyacrylamide gels were probed with monoclonal antibodies to α- and β-tubulin. At least six α- and seven β-tubulins were present in the extracts from both the nonacclimated and cold-acclimated roots. Changes in electrophoretic mobility and isotype number of both α- and β-tubulin were observed after only 2 days at 4°C. Further changes in tubulin were observed after 4 days at 4°C. Changes in α-tubulin were more pronounced than those in β-tubulin.     

Clostridium difficile Toxin A Decreases Acetylation of Tubulin,Leading to Microtubule Depolymerization through Activation of Histone Deacetylase 6, and This Mediates Acute Inflammation

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2′,3′-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.     

Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the α-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of α-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as α-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on α-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.     

Effects of α-Tubulin K40 Acetylation and Detyrosination on Kinesin-1 Motility in a Purified System

Long-range transport in cells is achieved primarily through motor-based transport along a network of microtubule tracks. Targeted transport by kinesin motors can be correlated with posttranslational modifications (PTMs) of the tubulin subunits in specific microtubules. To directly examine the influence of specific PTMs on kinesin-1 motility, we generated tubulin subunits that were either enriched in or lacking acetylation of α-tubulin lysine 40 (K40) or detyrosination of the α-tubulin C-terminal tail. We show that K40 acetylation does not result in significant changes in kinesin-1’s landing rate or motility parameters (velocity and run length) across experimental conditions. In contrast, detyrosination causes a moderate increase in kinesin-1’s landing rate. The fact that the effects of detyrosination are dampened by prior K40 acetylation indicates that the combination of PTMs may be an important aspect of the functional output of microtubule heterogeneity. Importantly, our results indicate that the moderate influences that single PTMs have on kinesin-1 in vitro do not explain the strong correlation between specific PTMs and kinesin-1 transport in cells. Thus, additional mechanisms for regulating kinesin-1 transport in cells must be explored in future work.     

Tubulin Tail Sequences and Post-translational Modifications Regulate Closure of Mitochondrial Voltage-dependent Anion Channel (VDAC)

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

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.