TGN46 is required for cargo sorting and loading into CARTS.

(A) PAUF secretion assay in parental (WT) and CRISPR/Cas9-mediated TGN46 knockout (KO) HeLa cells. Cells expressing PAUF-MycHis were incubated for 4 h in fresh medium without serum in the absence (Ctrl) or presence of Brefeldin A (BFA), after which the cells were lysed. Equal amounts of cell lysates and their corresponding secreted fraction (medium) were analyzed by western blotting (left) using an anti-Myc antibody (top blot) and anti-TGN46 antibody (bottom blot). BFA was included as a positive control of secretion inhibition. Quantification (right) of the ratio of secreted/internal PAUF signal normalized (norm.) as a percentage to the average value of the WT, Ctrl condition. n=4 independent experiments. Individual values shown with connecting lines between paired data sets (shown only for untreated condition, for clarity), with mean ± stdev. Paired t test (*, p ≤ 0.05). (B) Number of carriers (CARTS, empty circles; VSVG carriers, gray circles) per unit area observed in WT or TGN46-KO HeLa cells expressing either PAUF-mRFP (CARTS marker) and VSVG-HA (VSVG carrier marker). At least 10 cells from each of n=3 independent experiments were quantified. Individual values shown, with mean ± stdev. Unpaired two-tailed t test (ns, p > 0.05; *, p ≤ 0.05). (C) Relative fluorescence intensity average time trace (mean ± s.e.m.) of FLIP experiments performed in WT or TGN46-KO HeLa cells expressing PAUF-mRFP. (D) Quantification of the PAUF-mRFP Golgi residence time as obtained from the FLIP experiments, as the one shown in (C). Between 7–12 cells from each of n=3 independent experiments were quantified. Individual values shown, with mean ± stdev. Unpaired two-tailed t test (***, p ≤ 0.001). (E) Left panels are confocal fluorescence microscopy images of HeLa cells transfected with control (Ctrl) or TGN46 siRNA, which were also transfected with PKD2-KD-Flag and PAUF-mRFP. Cells were fixed at steady state and labelled with anti-TGN46 (cyan) and anti-Flag (magenta) antibodies. PAUF-mRFP fluorescence signal is shown in green. Scale bars are 10 µm, magnifications of the boxed regions are shown. In the right panel, the quantification of the percentage of PKD2-KD-induced tubules that containing PAUF-mRFP in control (Ctrl) or TGN46 siRNA-treated cells. At least 10 cells from each of n=3 independent experiments. Individual values shown, with mean ± stdev. Unpaired two-tailed t test (****, p ≤ 0.0001).

TGN46 export in CARTS is not dependent on cytosolic tail signals.

(A) Schematic representation of construct domain topology. Notice that type I proteins (e.g., GFP-TGN46) have a lumenal N-terminal domain, whereas type II proteins (e.g., ST-GFP) have a cytosolic N-terminal domain. TMD: transmembrane domain. (B) HeLa cells co-expressing the different indicated proteins (green and magenta channels) were fixed, and the localization of those proteins was monitored by fluorescence confocal microscopy. Insets correspond to zoom-in areas of the dashed, white boxed areas. (C) Pearson’s correlation coefficient between the perinuclear fluorescence signal of the x-axis indicated proteins with respect to TGN46-mRFP (empty circles) or ST-mCherry (gray circles), measured from confocal micrographs in (B). Results are from at least 10 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev). (D) HeLa 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. (E) 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 in (D). Results are from at least 10 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev). Scale bars in (B, D) are 10 µm.

FLIP experiments monitor Golgi residence times of different proteins.

(A, C, E–G) Relative fluorescence intensity average time trace (mean ± s.e.m.) of FLIP experiments for the indicated proteins. Symbols correspond to actual measurements, solid lines to the fitted exponential decays. (B, D) Residence time in the perinuclear area measured as the half time of the FLIP curves. Results are from 7–12 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev).

TGN46 intra-Golgi localization and CARTS specificity are insensitive to TMD length and composition.

(A) Schematic representation of construct domain topology. The amino acid sequence (in the correct topology) of the different transmembrane domains (TMD) is indicated. (B) HeLa cells co-expressing the different indicated proteins (green and magenta channels) were fixed, and the localization of those proteins was monitored by fluorescence confocal microscopy. Insets correspond to zoom-in areas of the dashed, white boxed areas. (C) Pearson’s correlation coefficient between the perinuclear fluorescence signal of the x-axis indicated proteins with respect to TGN46-mRFP (empty circles) or ST-mCherry (gray circles), measured from confocal micrographs in (B). Results are at least 3 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev). (D) HeLa 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. (E) 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 in (D). Results are from at least 10 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev). Scale bars in (B, D) are 10 µm.

Intra-Golgi localization analysis for the role of the lumenal domain of TGN46 in CARTS-mediated export from the TGN.

(A) Schematic representation of construct domain topology. TMD: transmembrane domain. (B) HeLa cells co-expressing the different indicated proteins (green and magenta channels) were fixed, and the localization of those proteins was monitored by fluorescence confocal microscopy. Insets correspond to zoom-in areas of the dashed, white boxed areas. (C) Pearson’s correlation coefficient between the perinuclear fluorescence signal of the x-axis indicated proteins with respect to TGN46-mRFP (empty circles) or ST-mCherry (gray circles), measured from confocal micrographs in (B). Results are from at least 10 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev). (D) HeLa 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. (E) Pearson’s correlation coefficient between the perinuclear fluorescence signal of the x-axis-indicated proteins with respect to TGN46-mRFP (empty circles) or VSVG-HA (gray circles; detected by immunofluorescence using an Alexa Fluor 647-conjugated secondary antibody), measured from confocal micrographs in (D). Results are from at least 10 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev). Scale bars in (B, D) are 10 µm.

The lumenal domain of TGN46 is necessary and sufficient for its CARTS-mediated export from the TGN.

(A) HeLa 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. (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 in (A). Results are from at least 10 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev).

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 PAUF (CARTS marker) as a function of the Golgi residence time as measured from 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 ± 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). (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 7–12 cells from each of n=3 independent experiments (individual values shown, with mean ± stdev).