A NOVEL 95-KD PROTEIN IS LOCATED IN A LINKER BETWEEN CYTOPLASMIC MICROTUBULES AND BASAL BODIES IN A GREEN FLAGELLATE AND FORMS STRIATED FILAMENTS IN-VITRO

The flagellar basal apparatus comprises the basal bodies and the attac hed fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isola ted from the naked green flagellate Spermatozopsis similis and shown t o be composed of several dozens of different polypeptides including a protein band of 95 kD, Screening of a cDNA library of S, similis with a polyclonal antibody raised against the 95-kD band resulted in a full -length clone coding for a novel protein of 834 amino acids (90.3 kD), Sequence analysis identified nonhelical NH2-and COOH-terminal domains flanking a central domain of similar to 50 residues, which was predic ted to form a series of coiled-coils interrupted by short spacer segme nts, Immunogold labeling using a polyclonal antibody raised against th e bacterially expressed 95-kD protein exclusively decorated the striat ed, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed f rom the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific tri plets of the basal bodies with the microtubular bundles that emerge fr om the basal apparatus. The sf-fibers and similar structures, e,g., ba sal feet or satellites, described in various eukaryotes including vert ebrates, may be representative for cytoskeletal elements involved in p ositioning of basal bodies/centrioles with respect to cytoskeletal mic rotubules and vice versa.