MOLECULAR-CLONING AND CHARACTERIZATION OF A RADIAL SPOKE HEAD PROTEINOF SEA-URCHIN SPERM AXONEMES - INVOLVEMENT OF THE PROTEIN IN THE REGULATION OF SPERM MOTILITY

Monoclonal antibodies raised against axonemal proteins of sea urchin s permatozoa have been used to study regulatory mechanisms involved in f lagellar motility. Here, we report that one of these antibodies, monoc lonal antibody D-316, has an unusual perturbating effect on the motili ty of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pat tern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE , D-316 recognized a single polypeptide of 90 kDa. This protein was pu rified following its extraction by exposure of axonemes to a brief hea t treatment at 40 degrees C. The protein copurified and coimmunoprecip itated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protei n sequences in databases indicated that the protein is related to radi al spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii , which share 37% and 25% similarity, respectively, with p63 However, the sea urchin protein possesses structural features distinct from RSP 4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results su ggest a major role for p63 in the maintenance of a planar form of sper m flagellar beating and provide new tools to study the function of rad ial spoke heads in more evolved species.