POSTTRANSLATIONAL MODIFICATIONS AND MULTIPLE TUBULIN ISOFORMS IN NICOTIANA-TABACUM-L CELLS

Distribution of post-translationally modified tubulins in cells of Nic otiana tabacum L. was analysed using a panel of specific antibodies. P olyglutamylated, tyrosinated, nontyrosinated, acetylated and Delta 2-t ubulin variants were detected on alpha-tubulin subunits; polyglutamyla tion was also found on beta-tubulin subunits. Modified tubulins were d etected by immunofluorescence microscopy in interphase microtubules, p reprophase bands, mitotic spindles as well as in phragmoplasts. They w ere, however, located differently in the various microtubule structure s. The antibodies against tyrosinated. acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodi es against nontyrosinated and Delta 2-tubulin provided dotlike stainin g of interphase microtubules. Additionally, immunoreactivity of antibo dies against acetylated and Delta 2-tubulins was strong in the pole re gions of mitotic spindles. High-resolution isoelectric focusing reveal ed 22 tubulin charge variants in N. tabacum suspension cells. Immunobl otting with antibodies TU-01 and TU-06 against conserved antigenic det erminants of alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit. Whereas antibodies against polyglutamylated. tyrosinate d and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and Delta 2-tubulin reacted with on ly one. The combined data demonstrate that plant tubulin is extensivel y posttranslationally modified and that these modifications participat e in the generation of plant tubulin polymorphism.