Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin

Abstract

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 epitopes 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 fusion peptides generated in E. coli to discriminate antibodies directed against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, using 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 alpha-tubulin; AXO 49 and a fraction of the PAT serum proved to be unreactive over fusion proteins; both PAT and AXO 49 labelled a restricted population of very stable microtubules in Paramecium, consisting of axonemal and cortical ones, and their reactivity was sequentially detected following microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, indicating the recognition of various alpha- and beta-tubulin isoforms displaying different apparent molecular masses. These data, taken as a whole, definitely establish that PAT and AXO 49 recognize a post-translational modification occurring in axonemal microtubules of protozoa as of metazoa. This modification appears to be distinct from the previously known ones, and all the presently available evidence indicates that it corresponds to the very recently discovered polyglycylation of Paramecium axonemal alpha- and beta-tubulin.