Posttranslational modifications of the C-terminus of alpha-tubulin in adult rat brain: alpha 4 is glutamylated at two residues.
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.
Eide Supérieure de Physique et Chimie Industrielles de la Ville de Paris, Laboratoire de Neurobiologie, CNRS UMR 7637, Paris, France.
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Redeker
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Rossier
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Frankfurter
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D007536:Isomerism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D010446:Peptide Fragments; D014404:Tubulin; D018698:Glutamic Acid; D014443:Tyrosine