The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.

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The relationship between microtubule dynamics and polyglutamylation of [3H]glutamate into a-and 3-tubulin, whereas taxol had no effect for a-tubulin and a stimulating effect for f-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of a-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of a-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (lst-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated a-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated a-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of a-and ,B-tubulin suggest that distinct enzymes are involved.