Phyloproteomics reveals conserved patterns of axonemal dynein methylation across the motile ciliated eukaryotes

Axonemal dynein assembly occurs in the cytoplasm and numerous cytosolic factors are specifically required for this process. Recently, one factor (DNAAF3/PF22) was identified as a methyltransferase. Examination of Chlamydomonas dyneins found they are methylated at substoichiometric levels on multiple sites, including Lys and Arg residues in several of the nucleotide-binding domains and on the microtubule-binding region. Given the highly conserved nature of axonemal dyneins, one key question is whether methylation happens only in dyneins from the chlorophyte algae, or whether these modifications occur more broadly throughout the motile ciliated eukaryotes. Here we take a phyloproteomic approach and examine dynein methylation in a wide range of eukaryotic organisms bearing motile cilia. We find unambiguous evidence for methylation of axonemal dyneins in alveolates, chlorophytes, trypanosomes, and a broad range of metazoans. Intriguingly, we were unable to identify a single instance of methylation on Drosophila melanogaster sperm dyneins even though dipterans express a Dnaaf3 orthologue, or in spermatozoids of the fern Ceratopteris, which assembles inner arms but lacks both outer arm dyneins and DNAAF3. Thus, methylation of axonemal dyneins has been broadly conserved in most eukaryotic groups and has the potential to variably modify the function of these motors.