Figure 7. The cargo sorting function of TGN46 is mediated by its lumenal domain.

(A) CARTS specificity of cargo proteins correlates with their Golgi export rate. Plot of the percentage of cargo-positive vesicles that are also positive for pancreatic adenocarcinoma upregulated factor (PAUF; CARTS marker) as a function of the Golgi residence time as measured from fluorescence loss in photobleaching (FLIP) experiments, for the different indicated cargo proteins (color coding explained in the legend on the right). Dashed black line represents a linear fit of the data points (shown as mean ± standard error of the mean [s.e.m.]), where the slope is statistically different from zero (extra sum-of-squares F test, p-value = 0.04). (B) Percentage of transport carriers containing each of the cargoes described on the x-axis that are also positive for PAUF (CARTS, empty circles) or VSVG (VSVG carriers, gray circles), as measured from confocal micrographs of HeLa cells (either WT or TGN46-KO cell lines) expressing the indicated proteins. Results are at least 10 cells from each of n = 3 independent experiment (individual values shown, with mean ± stdev; ns, p > 0.05; *p ≤ 0.05; ****p ≤ 0.0001). (C) Relative fluorescence intensity average time trace (mean ± s.e.m.) of FLIP experiments for the indicated proteins expressed in HeLa WT or HeLa TGN46-KO cells, as detailed in the legend. Symbols correspond to actual measurements, solid lines to the fitted exponential decays. (D) Residence time of PAUF-mRFP in the perinuclear area of HeLa cells (WT or KO), expressing the different proteins as labeled in the x-axis, and measured as the half time of the FLIP curves. Results are from 7 to 12 cells from each of n = 3 independent experiments (individual values shown, with mean ± stdev; ns, p > 0.05; **p ≤ 0.01).

Figure 7—figure supplement 1. The cargo sorting function of TGN46 is mediated by its lumenal domain.

(A) HeLa TGN46-KO cells co-expressing the different indicated proteins (green and magenta channels) were fixed, processed for immunostaining when required, and the localization of those proteins was monitored by fluorescence confocal microscopy. Insets correspond to zoom-in areas of the dashed, white boxed areas. Scale bars are 10 µm.

Figure 7—figure supplement 2. Golgi export of PAUF-mRFP is dependent on the lumenal domain of TGN46.

(A) Fluorescence microscopy images obtained from a characteristic fluorescence loss in photobleaching (FLIP) experiment assessing the export rate of PAUF-mRFP from the perinuclear area in HeLa cells (either WT or KO) expressing the indicated proteins. Time from the beginning of the FLIP experiments is indicated, and the area enclosed by the white lines shown in the left images denotes the photobleached area. Scale bars are 10 µm.

Figure 7—figure supplement 3. TGN46 domain structure and sequence characteristics.

(A) On the top, signaling peptide region (SP, gray) and mature TGN46 (TGN46, blue). On the bottom, the lumenal (lum, blue), transmembrane (TM, red), and cytoplasmic (cyt, green) domains. (B) Post-translational modifications. On the top, the glycosylation sites (purple) and on the bottom, the phosphorylation sites (yellow). (C) Disorder propensity indicated by PONDR score using the VL-XT algorithm (Li et al., 1999; Romero et al., 1997; Li et al., 1999). Values ranging 0.0–0.5 indicate order and values ranging 0.5–1.0 indicate disorder.