Fig. 3. Loss of binding to αβ-tubulins constitutively activates TTC5.

a Generated TTC5 constructs. b Indicated Strep-TTC5 constructs were expressed in HeLa TTC5 knockout cells and pulled down via Strep-tag. Bound αβ-tubulins were visualized by western blot. The asterisk indicates Strep-TTC5. The experiment was repeated three times with similar results. c BiFC approach. d Representative images of the indicated constructs expressed in HeLa TTC5 knockout cells. Scale bar = 20 µm. e Fluorescence intensity of Venus across the indicated BiFC cell lines. Red lines depict median and interquartile fluorescence intensities. Indicated are p-values in the two-sided Mann–Whitney test for each of the BiFC constructs with the one based on TTC5^WT as reference. The experiment was done with six biological replicates, analyzing 6465, 2005, and 5258 cells for WT, D175A, and T432E samples, respectively. f Sixty-four-residue α- and β-tubulin nascent chains (TUB NC) were produced in rabbit reticulocyte lysates in the presence of ³⁵S-methionine and recombinant WT or mutated Strep-TTC5. Strep-TTC5 and its associated proteins were subsequently enriched via the Strep-tag. Interacting partners were visualized by SYPRO Ruby staining, western blot, and autoradiography. β* indicates a β-tubulin construct in which its TTC5-interacting MREI motif has been mutated to autoregulation-incompatible MHQV. Data shown is representative of two replicate experiments for the D175A mutant, and from one experiment for the T432E mutant. Results were confirmed by orthogonal means (Fig. 3h). g Relative α- and β-tubulin mRNA levels in HeLa parental, TTC5 knockout, and the indicated Strep-TTC5 cell lines, normalized to a housekeeping transcript and the parental cell line. Data show the mean ± SD from three independent experiments. Indicated are p-values in unpaired, two-tailed Student’s t-tests for each of the indicated cell lines with the parental cell line as reference. h Autoregulation assay with HeLa parental, TTC5 knockout, and the indicated Strep-TTC5 cell lines. Data show the mean ± SD mRNA levels from three (D175A and T432E) and six (Parental, TTC5 KO and WT) independent experiments. Indicated are p-values in unpaired, two-tailed Student’s t-tests for each of the indicated cell lines with the DMSO-treated sample as reference. Source data for this figure are provided as a Source Data file.