Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly

Abstract

The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 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 RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.

Role of the Novel Hsp90 Co-Chaperones in Dynein Arms’ Preassembly

“…The phenotypic changes were caused by the reduction in the number of IDAs b and c, while IDAs f/I1 and g were almost unaffected or only slightly reduced, similarly to ODAs [51]. The biochemical studies revealed that MOT48 interacts with RuvBL1, RuvBL2, RPAP3, and DNAAF4/DYX1C (Table 1) [53].…”

“…To date, mutations in RuvBL1 or RuvBL2 have not been associated with PCD in humans, likely because these proteins function as co-chaperones for the assembly of diverse protein complexes and their mutations would cause death during early development, as was observed in mice [15]. [53] IDA c,d,g [52] DNAH5, DNAI1, [34] DNALI1 [34] DNAAF7 DNAH5, DNAI2 [58] TCTEX1D1 [58] DNAH5 [35] DNAL1 [35] LRRC6 DNAI1, DNAI2 [37,38] DNAH7 DNALI2 [37,38] Similarly, the analyses of Chlamydomonas and Danio rerio mutants suggested the involvement of PIH1D1 in dynein arms' assembly. Comparative studies of Danio rerio WT and pih1d1_null mutant revealed the abnormal motility of sperm flagella and Kupffer's vesicle cilia caused by the loss of outer and inner dynein arms.…”

“…The WD repeat-containing protein 92 (WDR92) has been reported to associate with the R2TP complex ( Figure 2) [59,60]. Recently, the participation of WDR92 and the canonical R2TP complex in the pre-assembly of dynein arms has been revealed [53][54][55]61]. The comparative genomic studies demonstrate that WDR92 genes are highly conserved in organisms with motile cilia [54,61].…”

“…The Chlamydomonas WRD92 mutant assembles very short cilia lacking inner and outer dynein arms. Moreover, the level of dynein arm heavy chains in the cytoplasm is low [53,54]. Data obtained during the comparative mass spectrometry analysis of wild type and Drosophila mutant sperm flagella [55], and analyses of the WDR92 Chlamydomonas phenotype [54], suggest that WDR92 is required for the stabilization of the folded dynein heavy chains (Table 2).…”

Rare Human Diseases: Model Organisms in Deciphering the Molecular Basis of Primary Ciliary Dyskinesia

“…To date, the data concerning interactions between DNAAFs, DNAAFs and dynein arms subunits, as well as DNAAFs and heat shock protein chaperones, are fragmentary. It is proposed that DNAAFs assist heat-shock protein chaperones in the folding and pre-assembly of these complexes [150,151].…”