| Using mutations that disrupt assembly of flagellar outer row dyneins in Chlamydomonas, we are exploring cytoplasmic steps necessary for axonemal dynein assembly. By immunoprecipitation, we show that outer row intermediate and light chains can assemble in the cytoplasm independently of heavy chains, and that first gamma, then beta and finally alpha heavy chains bind sequentially to this IC/LC core. Three loci that do not encode subunits of the motor disrupt assembly of outer row dynein in the cytoplasm, oda7, pf13 and pf22. Immunoblots indicate that these mutations affect the cytoplasmic stability of dynein heavy chains, and immunoprecipitation shows that they also disrupt co-assembly of heavy chains with intermediate chains. Although much more abundant in cytoplasm, ODA7 is also present in flagella and co-purifies with outer row and I1 dyneins. PF13 is a cytoplasmic protein weakly homologous to yeast PIH1, a co-chaperone, and our data suggests a co-chaperone func
tion for PF13 in dynein heavy chain folding/assembly. PF22 is a novel protein, and like PF13, PF22 is needed for assembly of some inner row dyneins as well as outer row dynein. All three assembly factors are conserved among organisms with motile cilia, and mutations in their vertebrate homologs result in Primary Ciliary Dyskinesia, suggesting that their functions have also been conserved. We are testing the hypothesis that one or more of these factors is important for the proper folding of dynein head domains. Supported by National Institutes of General Medical Sciences grant GM44228 to DRM. |