Multiple alpha-tubulin isoforms in cilia and cytoskeleton of Tetrahymena pyriformis generated by post-translational modifications. Studies during reciliation.
alpha-Tubulin microheterogeneity was studied in Tetrahymena pyriformis. Using two-dimensional electrophoretic analysis, we found between five and seven alpha-tubulin and four beta-tubulin isoforms in cilia and four or five alpha-tubulins and two beta-tubulins in cytoskeleton. Immunoblotting assay with anti-(acetyl alpha-tubulin) monoclonal antibody 6-11B-1 and [3H]acetate labelling revealed that the alpha-tubulin isoforms are post-translationally modified by acetylation. Our results also show that tubulins in the soluble cytoplasmic fraction are not acetylated. Nevertheless, a slight reaction with the antibody 6-11 B-1 can be observed in the taxol and vinblastine-treated cytoplasmic pool. Pulse/chase experiments using [35S]methionine during cell reciliation have demonstrated that the basic alpha-tubulin isoforms are converted into acidic isoforms in the absence of protein synthesis, suggesting that the basic alpha-tubulin is the precursor of the acidic forms which are found in cilia and cytoskeleton. In-vivo-translation selection demonstrated the existence of a single precursor molecule which corresponded to the most basic alpha-tubulin. Taken together, our results provide evidence for the existence of post-translational modifications, namely acetylation. Nevertheless, other post-translational mechanisms involved in the biosynthesis of microtubules of cilia and cytoskeleton are required to explain the whole alpha-tubulin heterogeneity.
Penque
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Laboratório de Genética Molecular, Instituto Gulbenkian de Ciência, Oeiras, Portugal.
Galego
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Rodrigues-Pousada
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D000085:Acetates / Q000378:metabolism; D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal; D002923:Cilia / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D014644:Genetic Variation; D007536:Isomerism; D008715:Methionine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D012038:Regeneration / Q000235:genetics*; D013462:Sulfur Radioisotopes; D013769:Tetrahymena pyriformis / Q000235:genetics*; D014316:Tritium; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics*
D000085:Acetates; D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D014404:Tubulin; D014316:Tritium; D008715:Methionine