Fig. 3. TBC1D23 binds to C-terminal conserved residues of the cytoplasmic tail of CPD.

(A) The cytoplasmic tail of human CPD. (B and C) Coomassie-stained gels of the eluates from immobilized GST-CPD truncations or mutants incubated with a bacterial lysate containing TBC1D23-His₆. Each representative of two repeats. (D) Volcano plot of the MS analysis from affinity chromatography of 293T cell lysates using bacterially expressed GST-TBC1D23 (559 to 684) or GST alone. Shown are mean spectral intensities of bound proteins from three independent experiments. Values are in data S1. (E) Coomassie-stained gel showing that the eluates from immobilized GST-TBC1D23 (559 to 684) incubated with lysates from bacteria expressing the indicated fragments of syntaxin-16 (STX16)–MBP–His₆ (numbering as in UniProt O14662-2). Representative of two repeats. (F) Cartoon of syntaxin-16 showing the location of the key structural features and the acidic TLY motif related to that of CPD. (G) Coomassie-stained gel showing that the eluates from immobilized GST-TBC1D23 (559 to 684) incubated with lysates from bacteria expressing His₆-MBP-CPD (1321 to 1380), syntaxin-16 (1 to 281)–MBP–His₆ or syntaxin-16 (1-281 with T191A, L192A, and Y193A)–MBP–His₆. Representative of two repeats. (H) Confocal micrographs of the indicated INS-1 cells (wild type, ∆TBC1D23, and ∆TBC1D23 stably expressing TBC1D23-GFP under a cumate promoter in the presence of cumate for 24 to 36 hours). Cells stained for GFP and endogenous syntaxin-16 and GM130. (I) Scatter plot (left) showing the ratio of the Golgi fluorescence intensity of syntaxin-16 over the GM130-positive regions (Golgi). The horizontal bar is the mean. For wild type, n = 530, for ∆TBC1D23, n = 628, and for the stable rescue, n = 725. ****P < 0.0001 [ordinary one-way analysis of variance (ANOVA) followed by a Sidak’s multiple comparison tests with a single pooled variance]. Ratios were also plotted as frequency distributions with a bin width of 0.05 (right). Values are in data S2.