Axonemal dyneins are highly complex molecular motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed for the generation of dynein holoenzymes and/or their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation which can generate degrons subject to the AcN-end rule and alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from Chlamydomonas to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex are processed to yield an unmodified Pro residue which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm. 

These data were generated from whole cilia and electrophoretically purified dynein heavy chains digested with trypsin or Asp-N using an Orbitrap Eclipse mass spectrometer and positive mode electrospray ionization. Data were searched against the Chlamydomonas v.6.1 proteome.