Tubulin Monoglutamylation is Sufficient to Rescue the Ciliary Motility Defects in a Chlamydomonas Polyglutamylation Deficient Mutant

Abstract

The axonemes of eukaryotic cilia and flagella display high tubulin glutamylation heterogeneity, yet the functional significance of this variation remains elusive. We previously showed that long-chain polyglutamylation is crucial for ciliary motility in Chlamydomonas. However, the respective contributions of long-chain polyglutamylation versus short-chain species to motility remain unclear, as existing mutants did not allow for a clear functional dissection of these two modification states. Here, we generated mutants deficient in deglutamylases, cytosolic carboxypeptidases (CCPs) 1, 2, and 5. Importantly, CCP5 is known to remove the branch-point glutamate residue, the final step in deglutamylation. While axonemal polyglutamylation levels remained largely unaffected in these mutants, abundance of short-chain glutamylation was significantly increased in both the axonemal and cytoplasmic microtubules of ccp5-1, consistent with CCP5s role as a branch-point deglutamylase. Although each single mutant exhibited slightly reduced swimming velocity, the loss of CCP5 in the tpg1 background lacking long polyglutamate side chains resulted in a significant restoration of motility. These findings indicate that the abundance of short-chain species, regulated by CCP5, plays a distinct role in modulating ciliary motility, particularly in the absence of long polyglutamate side chains. This suggests that even minimal glutamylation can functionally support dynein-driven microtubule sliding.