THE CHLAMYDOMONAS FLA10 GENE ENCODES A NOVEL KINESIN-HOMOLOGOUS PROTEIN

Many genes on the uni linkage group of Chlamydomonas affect the basal body/flagellar apparatus. Among these are five FLA genes, whose mutati ons cause temperature-sensitive defects in flagellar assembly. We pres ent the molecular analysis of a gene which maps to fla10 and functiona lly rescues the fla10 phenotype. Nucleotide sequencing revealed that t he gene encodes a kinesin-homologous protein, KHP1. The 87-kD predicte d KHP1 protein, like kinesin heavy chain, has an amino-terminal motor domain, a central alpha-helical stalk, and a basic, globular carboxy-t erminal tail. Comparison to other kinesin superfamily members indicate d striking similarity (64% identity in motor domains) to a mouse gene, KIF3, expressed primarily in cerebellum. In synchronized cultures, th e KHP1 mRNA accumulated after cell division, as did flagellar dynein m RNAs. KHP1 mRNA levels also increased following deflagellation. Polycl onal antibodies detected KHP1 protein in Western blots of purified fla gella and axonemes. The protein was partially released from axonemes w ith ATP treatment, but not with AMP-PNP. Western blot analysis of axon emes from various motility mutants suggested that KHP1 is not a compon ent of radial spokes, dynein arms, or the central pair complex. The qu antity of KHP1 protein in axonemes of the mutant fla10-1 was markedly reduced, although no reduction was observed in two other uni linkage g roup mutants, fla9 and fla11. Furthermore, fla10-1 was rescued by tran sformation with KHP1 genomic DNA. These results indicate that KHP1 is the gene product of FLA10 and suggest a novel role for this kinesin-re lated protein in flagellar assembly and maintenance.