Class I and IVa β-tubulin isotypes expressed in adult mouse brain are glutamylated

Several types of post-translational modifications contribute to the high level of tubulin heterogeneity in the brain. An important modification is glutamylation of the major brain-specific isotypes, such as class Ia/b of α-tubulin and classes II and III of β-tubulin. Here we describe experiments to determine if additional, minor tubulin isotypes, expressed in adult mouse brain, could also be glutamylated. Purified tubulin from adult mouse brain was cleaved with thermolysin. Proteolytically released carboxy-terminal peptides of both α- and β-tubulin were isolated by sequential anion exchange and reverse-phase column-chromatography. Anionic peptides were then characterized by amino acid sequencing and mass spectrometry. We show that brain-specific class IVa and constitutive class I β-tubulin isotypes can be glutamylated, at Glu434 and Glu441, respectively.