A Novel Basal Apparatus Protein of 90 kD (BAp90) from the Flagellate Green Alga Spermatozopsis similis is a Component of the Proximal Plates and Identifies the d-(dexter)Surface of the Basal Body

Abstract

The flagellar basal apparatus consists of the basal bodies and associated fibrous structures, and represents the organizing center for the microtubular cytoskeleton in many flagellate protists. To identify novel proteins associated with the basal bodies, basal apparatuses from the flagellate green alga Spermatozopsis similis (Chlorophyceae) were isolated and purified. A polyclonal antibody raised against a 95kD protein band enriched in purified basal apparatuses was used to screen a cDNA library of S. similis which resulted in the isolation of a full length clone coding for a novel protein of 812 amino acids (85.3kD). Sequence analysis of this clone identified extended a-helical regions and predicted several coiled-coil forming domains interrupted by spacer segments of variable lengths. A polyclonal antibody (anti-BAp90) raised against the bacterially expressed protein recognized a 90kD band (BAp90) in SDS-PAGE of isolated basal apparatuses of S. similis. Immunogold labeling using anti-BAp90 decorated the proximal plates (two striated, triangular fibers which serve as spacers between the basal bodies in their proximal region) and parts of the d-fibers (df) which interconnect the basal bodies with the microtubular d-roots and the striated microtubule-associated fibers (SMAFs). Thus, the 90kD basal apparatus protein of S. similis represents a biochemical landmark for the lateral asymmetry of the basal body identifying its d-(dexter)surface. Cytoskeletal elements containing BAp90 or structurally related proteins may be involved in the organization and/or maintenance of the positional relationship between basal bodies and the cellular cytoskeleton, and hence cell polarity.