MONOCLONAL AND POLYCLONAL ANTIBODIES DETECT A NEW-TYPE OF POSTTRANSLATIONAL MODIFICATION OF AXONEMAL TUBULIN

Polyclonal (PAT) and monoclonal (AXO 49) antibodies against Paramecium axonemal tubulin were used as probes to reveal tubulin heterogeneity. The location, the nature and the subcellular distribution of the epit opes recognized by these antibodies were, respectively, determined by means of: (i) immunoblotting on peptide maps of Paramecium, sea urchin and quail axonemal tubulins; (ii) immunoblotting on ciliate tubulin f usion peptides generated in E, coli to discriminate antibodies directe d against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, usin g throughout an array of antibodies directed against tubulin sequences and post-translational modifications as references. AXO 49 monoclonal antibody and PAT serum were both shown to recognize epitopes located near the carboxyl-terminal end of both subunits of Paramecium axonemal tubulin, whereas the latter recognized additional epitopes in a-tubul in; AXO 49 and a fraction of the PAT serum proved to be unreactive ove r fusion proteins; both PAT and AXO 49 labelled a restricted populatio n of very stable microtubules in Paramecium, consisting of axonemal an d cortical ones, and their reactivity was sequentially detected follow ing microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, ind icating the recognition of various alpha- and beta-tubulin isoforms di splaying different apparent molecular masses. These data, taken as a w hole, definitely establish that PAT and AXO 49 recognize a post-transl ational modification occurring in axonemal microtubules of protozoa as of metazoa, This modification appears to be distinct from the previou sly known ones, and all the presently available evidence indicates tha t it corresponds to the very recently discovered polyglycylation of Pa ramecium axonemal alpha- and beta-tubulin.