Purification and characterization of basal apparatuses from a flagellate green alga

Abstract

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis similis by detergent extraction and mechanical disintegration. Sucrose density centrifugation yielded highly enriched basal apparatuses as shown by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated basal apparatuses. A mass spectrometric analysis of the carboxyterminal peptides of α tubulin documented detyrosination and glutamylation as posttranslational modifications and showed that some 5% of the α 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 basal apparatus. A 210-kD component identified by mAB BAS (basal apparatus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted with two high molecular weight bands (≈ 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end of the basal bodies. Immunofluorescence staining of isolated cytoskeletons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardii and Dunaliella bioculata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus. Cell Motil. Cytoskeleton 37:72–85, 1997. © 1997 Wiley-Liss, Inc.