PURIFICATION AND CHARACTERIZATION OF BASAL APPARATUSES FROM A FLAGELLATE GREEN-ALGA

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis si milis by detergent extraction and mechanical disintegration. Sucrose d ensity centrifugation yielded highly enriched basal apparatuses as sho wn by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated bas al apparatuses. A mass spectrometric analysis of the carboxyterminal p eptides of alpha tubulin documented detyrosination and glutamylation a s posttranslational modifications and showed that some 5% of the alpha tubulin carries a polyglutamyl side chain which can reach at least 17 residues in length. Monoclonal antibodies raised against the purified basal apparatuses were used to characterize novel components in the b asal apparatus. A 210-kD component identified by mAB BAS (basal appara tus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted w ith two high molecular weight bands (approximate to 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end o f the basal bodies. Immunofluorescence staining of isolated cytoskelet ons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardtii and Dunaliella bio culata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus. (C) 1997 Wiley-Liss, Inc.