A combination of posttranslational modifications is responsible for the production of neuronal alpha-tubulin heterogeneity.

We describe the presence of alpha-tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyme(s) copurified with microtubules through two cycles of assembly-disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both alpha-tubulin and MAP2. To determine the site of the modification, tubulin was purified and digested with Glu-C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained epsilon N-acetyllysine at position 40 of the alpha-tubulin molecule. This result demonstrates that mouse brain alpha-tubulin was acetylated at the same site as in Chlamydomonas. Isoelectric focusing analysis showed that acetylated alpha-tubulin was resolved into five isoelectric variants, denoted alpha 3 and alpha 5 to alpha 8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys40 of an heterogeneous population of alpha-tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal alpha-tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.