View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by MPG.PuRe REVIEWS POST-TRANSLATIONAL MODIFICATIONS REGULATE MICROTUBULE FUNCTION Stefan Westermann* and Klaus Weber ‡ The αβ-tubulin heterodimer, the building block of microtubules, is subject to a large number of post-translational modifications, comparable in diversity to the intensively studied histone modifications. Although these unusual modifications are conserved throughout evolution, their functions have remained almost completely elusive. Recently, however, important advances in the understanding of how tubulin modifications regulate function and organization have been made. αβ MITOTIC SPINDLE Microtubules are an important component of the domain of -tubulins, which is located on the outside 3 A bipolar array of microtubules cytoskeleton and carry out a variety of essential func- of the microtubule where it is well positioned to influ- that functions to move the tions. During cell division, microtubules form the ence interactions with other proteins. duplicated chromosomes MITOTIC SPINDLE, the structure that is required to faith- Here,we review the literature on the occurrence of during mitosis and meiosis. fully segregate replicated sister chromatids. Together these modifications, the enzymes that are involved in AXONEME with accessory proteins, they constitute the AXONEME of generating them, and their potential functions. A bundle of microtubules and CILIA and FLAGELLA and so contribute to cell motility. In associated proteins that form the addition, they are important factors in the generation The role of the tubulin tails core of a flagellum or cilium. of cell polarity and also function as tracks along which Electron crystallographic studies of the αβ-tubulin CILIA — with the help of motor proteins — organelles and dimer have provided important insights into its struc- Hair-like extensions of cells, vesicles are transported through the cell. This diversity ture. However, they failed to resolve the carboxy-terminal which contain a microtubular of roles raises the question of how individual ten residues of α-tubulin and the carboxy-terminal 18 axoneme. Beating movements of microtubules, which are formed by polymerization residues of β-tubulin4.These residues immediately fol- cilia are responsible for of αβ-tubulin heterodimers (BOX 1),are assigned to low helices H11 and H12 in the dimeric tubulin struc- swimming. these specific functions. In other words, what distin- ture and are thought therefore to be located on the guishes a spindle microtubule from one that is part outer surface of the microtubule (FIG. 1).The failure to of a cilium? One answer is that the cell generates resolve these residues in the crystal structure could be microtubule diversity on two different levels1. First, due to the fact that they are disordered, as indicated by various different α- and β-tubulin isotypes are expressed some structural studies on synthetic carboxy-terminal 5 *Department of Molecular within the cell. However, although some tubulin iso- peptides .Not only are the carboxy-terminal sequences and Cell Biology, University types clearly have specific functions, in most cases that of α- and β-tubulin highly acidic, but they also consti- of California, Berkeley, have been studied, the isoforms seem functionally inter- tute the ‘isotype defining regions’6,where the various California 94720, USA. ‡ changeable. Second, microtubule diversity is also gener- tubulin isotypes differ most strongly from each other. Max Planck Institute for αβ Biophysical Chemistry, ated by an extensive array of reversible post-translational Several studies indicate that -tubulin that lacks Department of Biochemistry, modifications2,such as acetylation, polyglycylation and the carboxy-terminal domains can still polymerize into Am Fassberg 11, 37077 polyglutamylation, tyrosination/detyrosination, phos- microtubules, and that these microtubules are, in fact, Goettingen, Germany. phorylation and palmitoylation (TABLE 1).In fact, for more stable than wild-type microtubules7,8.However,it Correspondence to S.W. some PROTISTS that express only a single set of αβ-tubulin is possible that these domains might be important for e-mail: swesterm@uclink. berkeley.edu genes, this is the only source of tubulin diversity. Most the generation of higher-order microtubule structures doi:10.1038/nrm1260 of these modifications affect the carboxy-terminal — for example, a testis-specific β2-tubulin variant that 938 | DECEMBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio © 2003 Nature Publishing Group REVIEWS reminiscent of post-translational modifications of the Box 1 | The tubulin family amino-terminal histone tails11,which also lie on the outside of a polymer (in this case, chromatin), where 13 they are accessible to modifying enzymes and can interact with other proteins to alter the properties of 25 nm the polymer. Acetylation/deacetylation of α-tubulin When does it occur? Most α-tubulins are known to be acetylated on the ε-amino group of a conserved lysine 12,13 αβ residue at position 40 in the amino terminus . Acetylation is mostly associated with stable microtubu- lar structures such as axonemes, and it occurs after microtubule assembly14. On the basis of the electron- αβ heterodimers Short protofilaments Microtubule crystallographic structure, it has been predicted that the modified residue points towards the lumen of the 3 α- and β-tubulin form a heterodimer of 110 kDa, and head-to-tail association of these microtubule .Acetylation occurs in the primitive 15 16 dimers leads to the formation of linear protofilaments. The lateral association of eukaryotes Giardia lamblia , Tritrichomonas mobilensis protofilaments — usually 13 — forms a cylindrical microtubule with an outer diameter and Trypanosoma brucei17, and therefore arose early dur- of 25 nm. Microtubules are inherently polar structures with a dynamic plus end and a ing eukaryotic evolution. minus end that can be stabilized by embedding the filament into a microtubule- organizing centre (MTOC). The dynamic behaviour of individual microtubules in vivo is Which enzymes are required? A partial purification of a described by the term ‘dynamic instability’,as a microtubule can frequently switch tubulin acetyltransferase activity from CHLAMYDOMONAS between phases of growth and rapid depolymerization. The chemical basis for this flagella has been reported18,but the enzyme that is behaviour is thought to be a delayed hydrolysis of GTP at the β-subunit, which creates a responsible has not been identified. By contrast, two GTP-cap that promotes further microtubule growth. enzymes that catalyse the opposing reaction — that is, Structural similarities between tubulin and the bacterial cell-division protein FtsZ deacetylation — have been recently described19–21: 98 indicate that these proteins have a common ancestor .Further members of the tubulin HDAC6 (histone deacetylase 6) is a member of the γ family include -tubulin, a protein for which a role in nucleating microtubules is well histone deacetylase family that is predominantly cyto- ∆ ε ζ η established. Four ‘new’ tubulins — delta ( )-, epsilon ( )-, zeta ( )- and eta ( )-tubulin plasmic and colocalizes with microtubules. It can 99 — have been discovered recently . The conspicious absence of these proteins from deacetylate tubulin and microtubules in vitro, and sup- organisms that lack bona fide centrioles, together with a functional characterization of pression of HDAC6 function by pharmacological ε-tubulin100,strongly indicate that the new tubulins have functions that are associated inhibitors or by small interfering RNA (siRNA) in vivo with the eukaryotic centriole and/or basal body. So far, the post-translational leads to a marked increase in tubulin acetylation. modifications discussed in this review have only been described for α- and β-tubulins. γ-tubulin, as well as the new tubulins, generally lacks the highly acidic carboxy-terminal Conversely, overexpression of HDAC6 greatly decreased target sequences. the level of tubulin acetylation. A second histone Many eukaryotic cells express various α- and β-tubulin isotypes. In vertebrates, six deacetylase, SIRT2 (the human Sir2 orthologue), which + different genes for each α- and β-tubulin form a multigene family. Within one species, the is NAD dependent, has also been identified as a tubulin 22 greatest sequence variability between tubulin isotypes is found in the carboxy-terminal deacetylase , and shows preferential activity towards a tails (isotype-defining sequences). tubulin peptide substrate in comparison to a histone pep- tide substrate. SIRT2 co-immunoprecipitates with lacks the carboxy-terminal domain fails to assemble into HDAC6 from cell extracts, which raises the possibility that 9 FLAGELLA afunctional axoneme in Drosophila melanogaster .In these enzymes cooperate to deacetylate tubulin in vivo. Long protrusions that contain a addition, elegant genetic studies in TETRAHYMENA have microtubular axoneme, the recently addressed the role of the tubulin tails10.Tail Functions of microtubule acetylation. Microtubules beating of which can drive a cell α β through liquid media. Note that deletions of either - or -tubulin proved to be acetylated with partially purified Chlamydomonas bacterial flagella are constructed lethal. However, the essential role of both tails is tubulin acetyltransferase showed normal assembly/dis- very differently from eukaryotic redundant because, in chimeric tubulins, the car- assembly characteristics