SUBPELLICULAR AND FLAGELLAR MICROTUBULES OF TRYPANOSOMA-BRUCEI ARE EXTENSIVELY GLUTAMYLATED

To determine the spectrum of tubulin variants in cytoskeletons of Tryp anosoma brucei carboxy-terminal fragments of alpha- and beta-tubulin w ere isolated and characterized by sequencing and mass spectrometry. Al l variants arise by posttranslational modifications. We confirm the pr esence of tyrosinated and detyrosinated alpha-tubulin, Unexpectedly, b ut in line with its sequence, beta-tubulin also occurs with and withou t its carboxy-terminal tyrosine. Both tyrosinated and detyrosinated al pha- and beta-tubulins are extensively glutamylated. Unglutamylated tu bulins are only trace components of the cytoskeletal microtubules, The maximal numbers of glutamyl residues in the lateral chain are 15 and 6 for alpha- and beta-tubulin, respectively. The oligoglutamyl side ch ain is linked via an isopeptide bond to glutamic acid residues 445 of alpha- and 435 of beta-tubulin. The same sites are used in glutamylate d tubulins of mammalian brain, No tubulin variants based on polyglycyl ation are detected in cytoskeletal preparations or in isolated flagell a. Tubulin specific incorporation of radioactive glutamate but not of glycine is observed when protein biosynthesis is completely inhibited in Trypanosoma cells. Possible reasons for the absence of polyglycylat ed tubulins from the trypanosomal axoneme are discussed, Finally we sh ow that lysine 40 of the flagellar alpha-tubulin is completely acetyla ted.