Function Of Chlamydomonas WDR92 Protein In Dynein Arm Pre-Assembly

Eukaryotic cilium(or flagellum),composed of ciliary membrane,ciliary substrate and ciliary axonemal,is a kind of conserved organelle that protrudes from the cell surface.The cilium is powered by the axonemal dyneins,which are pre-assembled in the cytoplasm by proteins termed dynein arm assembly factors(DNAAFs)before being transported to and assembled on the ciliary axoneme,however,the detail molecular mechanism remains illusive.R2TP complex is a HSP90 co-chaperone and is involved in the assembly of large protein complexes including RNA polymerase II.WDR92 is a R2TP complex associated protein regulating the cell apoptosis,which has been reported functioning in ciliary microstructure assembly including dynein arms recently.In this article,we attempt to focus on WDR92 and characterize its function in Chlamydomonas to explore the molecular mechanism of DNAAFs regulating dynein arm assembly.Loss of WDR92,a cytoplasmic protein,in a wdr92 mutant generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella,disappearance of axonemal dynein arms,and diminishment of dynein arm heavy chains in the cytoplasm,suggesting that WDR92 is a DNAAF.Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including Ruv BL1,RPAP3,MOT48,ODA7,and DYX1 C.PIH proteins play indispensable roles in various biological processes,including forming the R2TP complex and regulating the cilia motility.There are three PIH1domain-containing proteins,MOT48,PF13 and TWI1 existing in Chlamydomonas.MOT48 formed a R2TP-like complex with Ruv BL1/2 and RPAP3 in vivo,while PF13,another PIH protein with function in dynein pre-assembly,did not.Interestingly,the third PIH protein TWI1 was not related to ciliary motility.WDR92 physically interacted with the R2TP-like complex and the other identified DNAAFs.Our data suggested that WDR92 functions in association with the HSP90 co-chaperone R2TP complex as well as linking other DNAAFs in axonemal dyneins pre-assembly.A variety of DNAAFs besides WDR92 regulate the functions of R2TP complex.We obtained an fbb18 mutant,whose phenotypes are similar to wdr92 mutant.The research about FBB18 suggested that FBB18 interacts with Ruv BL1/2 physically,and influences the protein level of R2TP-like complex.Further more,PF22,as another DNAAF,interacts with MOT48 in vitro and regulates the stability of MOT48 and RPAP3.All of these data support our opinion that DNAAFs regulate dynein arm pre-assembly in cooperation with the core R2TP complex.