The tubulin poly-glutamylase complex (TPGC) is comprised of TTLL1 and at least five associated proteins that promote the addition of glutamate residues to carboxy-terminal tails of tubulin subunits comprising microtubules. Despite its discovery two decades ago, the enzyme has been refractory to characterization owing to its complex multimeric nature and the inability to detect poly-glutamylase activity after assembling the six-subunit complex. We now show that TPGC is the key enzyme driving centriolar and ciliary poly-glutamylation. We identified two additional TPGC subunits, TBC1D19 and KIAA1841, and showed that both components play an essential role in the assembly of the eight-subunit holo-enzyme. We were able to reconstitute the activity of TPGC with all eight subunits. TBC1D19 and KIAA1841 were essential for TPGC activity, and loss of TBC1D19 strongly compromised multiple tubulin modifications, including axonemal poly-glutamylation. TBC1D19 loss also abolished transport of ARL13B and other ciliary membrane proteins, abrogating primary cilium assembly. Structural predictions suggested an essential role for TBC1D19 and KIAA1841 in complex assembly, microtubule binding, and preferential poly-glutamylation of α-tubulin. We found that TBC1D19 loss abrogated the ciliary localization of phosphatidyl inositol phosphatase, INPP5E, which coincided with cilium instability. Ciliogenesis in TBC1D19 null cells could be restored through inhibition of a specific phosphatidyl inositol phosphate (PIP) kinase, PIP5K1C, suggesting that TBC1D19 is required to maintain PIP homeostasis during ciliogenesis. Collectively, our data show that TPGC is a multi-functional enzyme essential for cilium assembly and maintenance.
Badarudeen et al. (Fri,) studied this question.