Biochemistry zyxwvu 1992, 31, zyxwvu 403-410 403 Polyglutamylated a-Tubulin Can Enter the Tyrosination/Detyrosination Cycle? Bernard Eddi,*+' Jean Rossier,§ Jean-Pierre Le Caer,§ Jean-Claude Promi,II Elisabeth DesbruyEres,f Franqois Gros,t and Philippe Denoulett Laboratoire de Biochimie Cellulaire, Coll2ge de France, zyxwvu I1 Place Marcelin Berthelot, 75231 Paris Cedex 05, France, Laboratoire de Physiologie Nerveuse, CNRS, 91 I98 Gif sur Yvette, France, and Centre de Recherche de Biochimie et GtnPtique Cellulaire, CNRS, 1 I8 Route de Narbonne, 31 062 Toulouse, France Received July zyxwvutsr 16, 1991; Revised Manuscript Received October 10, 1991 ABSTRACT: We have previously identified a major modification of neuronal a-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the y-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated a-tubulin [Eddb, B., Rossier, J., Le Caer, J. P., DesbruyEres, E., Gros, F., zyxwv & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated a-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six a-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of a-tubulin, the structure of this region was investigated. [3H]tyr~~inated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides cor- responds to the native C-terminal part of a-tubulin 440VEGEGEEEGEEY45' and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated a-tubulin. A quantitative analysis showed that the different tyrosinated forms of a-tubulin represent a minor (13%) fraction of the total a-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated. The different forms of a-tubulin were found to be equal substrates for tubulin tyrosine ligase and tubulin carboxypeptidase, which indicates that a-tubulin can enter the tyrosination/detyrosination cycle independently of its degree of glutamylation. Tubulin heterogeneity is strikingly high in the brain, where it is essentially due to the contribution of neuronal cells (Gozes & Littauer, 1978; Dah1 & Weibel, 1979; Gozes & Sweadner, 1981; George et al., 1981; Denoulet et al., 1982; Wolff et al., 1982; Moura-Net0 et al., 1983; Field & Lee, 1988). This heterogeneity is generated at two levels: differential expression of several isotypes which are encoded by different isogenes (Lewis et al., 1985; Villasante et al., 1986; Sullivan & Cleveland, 1986) and extensive posttranslational modifications of some, if not all, primary gene products. Several modifi- cations of a-tubulin have so far been characterized: removal and possible readdition of the C-terminal tyrosine (Barra et al., 1974), acetylation of K40 (L'Hernault & Rosenbaum, 1985; Le Dizet & Piperno, 1987; EddB et al., 1991), and polyglutamylation of E445 (Eddb et al., 1990). This latter modification consists of the progressive addition of a various ' number of glutamyl units forming a peptidic or pseudo-peptidic lateral chain extending from the main chain at a few residues from the C-terminus. Posttranslational modifications of p- tubulin are yet largely unknown, but phosphorylation (Edd6 et al., 1981; Gard & Kirschner, 1985) and polyglutamylation (Alexander et al., 1991) of a class I11 0-tubulin isotype have been described. tThis work was supported by grants from Centre National de la Recherche Scientifiqut (URA 11 15 and UPR 2212), Institut National de la SantE et de la Recherche MEdicale (CRE zyxwvutsr 89.6005), Commission of the European Communities (Cl.l-0508-FR-H), and Fondation pour la Recherche MEdicale Frangaise. * To whom correspondence should be addressed. 8 Laboratoire de Physiologie Nerveuse, CNRS. 11 Centre de Recherche de Biochimie et GEnEtique Cellulaire, CNRS. Laboratoire de Biochimie Cellulaire, Collbge de France. High-resolution IEF analysis has shown that neuronal a- tubulin is composed of as many as eight isoelectric variants. Among these, the most basic variant, denoted a l , corresponds to the primary products of several isogenes [mal, ma2, and ma4 in the mouse (Lewis et al., 1985; Villasante et al., 1986)], whereas the seven other more acidic forms are produced by posttranslational modifications (Denoulet et al., 1986, 1988; EddB et al., 1989). This isoelectric heterogeneity could be accounted for by polyglutamylation, acetylation, and probably other yet unknown modifications. In contrast to glutamylation and acetylation, tyrosination/detyrosination does not affect the net charge of the polypeptide although it can also con- tribute to tubulin diversity. This modification takes place at a site located very close to the site of glutamylation, raising the possibility that the two modifications could interfere with each other. This question is of fundamental importance, es- pecially as an abundant fraction of brain tubulin does not undergo the tyrosination/detyrosination cycle (Rodriguez & Borisy, 1978; Paturle et al., 1989). In a previous study (Eddb et al., 1990), we posttransla- tionally labeled tubulin with a radioactive precursor of glu- tamate and isolated polyglutamylated C-terminal peptides which were mainly in the detyrosinated form, although some peptides were also detected which could correspond to tyro- sinated forms. In this report, tyrosinated a-tubulin, post- translationally labeled by incubating cultured neurons with I Abbreviations: m/z, mass/charge ratio; PTH, phenylthiohydantoin; TTL, tubulin tyrosine ligase; HPLC, high-performance liquid chroma- tography; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing. Amino acids are indicated with the one-letter code: K, lysine; G, glycine; E, glutamate; Y, tyrosine; V, valine. 0006-2960/92/0431-403$03.00/0 0 1992 American Chemical Society