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Identification of a component of the Tubulin Poly-glutamylase Complex required for phosphoinositide homeostasis and cilium assembly and maintenance

ABSTRACT: 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 alpha-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. This entry contains the raw mass spectrometric data, methods and results for the affinity purification of TBC1D19.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Beatrix M Ueberheide

PROVIDER: MSV000100885 | MassIVE | Wed Feb 18 11:58:00 GMT 2026

SECONDARY ACCESSION(S): PXD074602

REPOSITORIES: MassIVE

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Project description:The essential process of dosage compensation equalizes X-chromosome gene expression between C. elegans XO males and XX hermaphrodites through a dosage compensation complex (DCC) that resembles condensin. The DCC binds to both X chromosomes of hermaphrodites to repress transcription by half. Here we show that post-translational modification by the SUMO conjugation pathway is essential for sex-specific assembly of the DCC onto X. Depletion of the SUMO peptide in vivo severely disrupts binding of particular DCC subunits and causes changes in X-linked gene expression similar to those caused by disrupting genes encoding DCC subunits. Three DCC subunits are themselves SUMOylated, and depletion of SUMO preferentially reduces their binding to X, suggesting that SUMOylation of DCC subunits is essential for robust association with X. DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly--binding of X-targeting factors to recruitment sites on X (rex sites)--is independent of SUMOylation, but robust binding of the complete complex requires SUMOylation. SUMOylated DCC subunits are enriched at rex sites, and SUMOylation enhances interactions between X-targeting factors and condensin subunits that facilitate DCC binding beyond the low level achieved without SUMOylation. DCC subunits also participate in condensin complexes essential for chromosome segregation, but their SUMOylation occurs only in the context of the DCC. Our results reinforce a newly emerging theme in which multiple proteins of a complex are SUMOylated in response to a specific stimulus, leading to accelerated complex formation and enhanced function. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, DPY-28 and FLAG-tagged SDC-2 in wild-type and smo-1 RNAi treated mixed embryos, with IGG controls. Also, sequential ChIP-chip experiments: (1) ChIP using FLAG antibodies to determine the genome-wide binding sites for SUMOylated proteins, (2) ChIP using FLAG antibodies followed by re-ChIP of eluted protein-chromatin complexes with DPY-27 antibodies to determine genome-wide binding sites for SUMOylated DPY-27 (referred to as DPY-27 re-ChIP experiments), (3) ChIP using FLAG antibodies followed by re-ChIP of eluted protein-chromatin with IGG antibodies to determine background binding (referred to as IGG re-ChIP experiments, and (4) ChIP using DPY-27 antibodies as a control to assess the efficiency of DPY-27 binding and detection in control vs. FLAG-tagged strains.

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Identification of a component of the Tubulin Poly-glutamylase Complex required for phosphoinositide homeostasis and cilium assembly and maintenance

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