Abstract

In adult mammalian brain, the C-terminus of alpha-tubulin exhibits a high degree of polymorphism due to a combination of four covalent posttranslational modifications: glutamylation, tyrosination, detyrosination, and removal of the penultimate glutamate residue (C-terminal deglutamylation). Glutamylation is the most abundant. To characterize the glutamylation of alpha-tubulin and its relationship with the other modifications, we developed a chromatographic procedure for purifying alpha-tubulin C-terminal peptides. The purified peptides were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and amino acid sequencing. In this report, we provide a complete description of the glutamylation of tyrosinated, detyrosinated, and C-terminal deglutamylated isoforms of both alpha-tubulin isotypes (alpha1/2 and alpha4) expressed in adult rat brain. In particular, we describe for the first time the glutamylation of alpha4. More than 90% of the alpha-tubulin is glutamylated, and more than 75% of it is nontyrosinated. alpha4 is more extensively glutamylated than alpha1/2, containing as many as 11 posttranslationally added glutamate residues. The most abundant alpha4 isoform is nontyrosinated, containing five posttranslationally added glutamates, whereas the most abundant alpha1/2 isoforms are nontyrosinated, with only one or two posttranslationally added glutamates. In contrast to alpha1/2, alpha4 is glutamylated at two separate residues (Glu-443 and Glu-445) in the sequence 431DYEEVGIDSYEDEDEGEE448. This is the first evidence that glutamylation can occur on two different residues in the same mammalian tubulin isotype.