PF20 GENE-PRODUCT CONTAINS WD REPEATS AND LOCALIZES TO THE INTERMICROTUBULE BRIDGES IN CHLAMYDOMONAS FLAGELLA

The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecu lar analysis of these components, we generated central apparatus-defec tive mutants in Chlamydomonas reinhardtii using insertional mutagenesi s. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed fl agella, and the entire central apparatus is missing in isolated axonem es. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued tra nsformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid s equence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate c hains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates t hat the PF20 protein is localized along the length of the C2 microtubu le on the intermicrotubule bridges connecting the two central microtub ules. We suggest that the PF20 gene product is a new member of the fam ily of WD repeat proteins and is required for central microtubule asse mbly and/or stability and flagellar motility.