ASSEMBLY OF FLAGELLAR RADIAL SPOKE PROTEINS IN CHLAMYDOMONAS - IDENTIFICATION OF THE AXONEME BINDING DOMAIN OF RADIAL SPOKE PROTEIN-3

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. I n the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in t he gene for one of 17 polypeptides that comprise the radial spokes res ults in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes . In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The so le axoneme binding domain of RSP3 is located within amino acids 1-85 o f the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated p rotein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its f unction, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.