Figure 6. Syn5-GPP130 interaction is required for STxB retrograde trafficking.

(a) Retrograde trafficking of STxB in scrambled (-) or siGPP130 (+) treated cells. Where indicated, cells were transfected with siRNA resistant wild-type GPP130_1-108 (WT), or the KR/AA-mutant of GPP130_1-108. Quantification of STxB-Cy3 labeling in the Golgi area in the indicated conditions. Graph shows mean +/- SD for 52 (control), 47 (siGPP130), 39 (WT rescue), 29 (KR, AA rescue) cells from 2 out of 3 independent experiments. Each point represents a cell. One-way ANOVA was performed overall (P < 0.0001) with Sidak’s multiple comparisons test. NS P = 0.8903, *** P < 0.0001. (b) Retrieval of GPP130 from endosomes. Gene-edited cells lacking GPP130 were transfected with either HA-GPP130_1-108 (WT) or the KR/AA-GPP130 mutant construct. Cells were then harvested before treatment (pre-treatment), treated with monensin for 1 h to redistribute GPP130 to endosomes, or monensin-treated and then subjected to a 3 h washout. Cells were labeled with GPP130 (green) and giantin (red). Representative immunofluorescence images are shown from 9 independent experiments. Dotted lines indicate nuclei, dashed lines show areas of zooms. Scale bars = 10 μm. (c) Quantification of number of cells with GPP130 primarily Golgi-localized, a mix of Golgi- and endosome-localized, or primarily endosome-localized, in washout conditions for WT and KR-AA transfected cells in (b). Graph shows mean +/- SD of ~50 cells from 9 independent experiments. Two-way ANOVA was performed overall (P < 0.0001) with Sidak’s multiple comparisons test. Golgi: ** P = 0.0044, Both: NS P = 0.9925, Endosomes ** P = 0.0027. (d) Redistribution of GPP130 in Retro-2, siSec16A, or siSar1A/B conditions. Confocal microscopy was performed for GPP130 (red), Giantin (green), EEA1 (blue in right panels) and Hoechst (blue in left panels). Dotted lines indicate zoomed areas. Arrows indicate GPP130 spots colocalizing with EEA1-positive vesicles. Scale bars = 10 μm. Representative images from 3 independent experiments. (e) Quantification of GPP130 in Golgi area as defined by giantin. Graph shows mean +/- SD for 31 (Scrambled), 29 (Retro-2), 27 (siSec16A), 29 (siSar1A/B) cells from 2 out of 3 independent experiments. Each point represents one cell. One-way ANOVA performed overall (P < 0.0001) with Dunnett’s multiple comparisons test comparing all conditions to scrambled, Retro-2: ** P = 0.0044, siSec16A/siSar1A/B: *** P < 0.0001. (f) Quantification of GPP130 in EEA1 positive structures. Graph shows mean +/- SD for 35 cells per condition from 2 out of 3 independent experiments. Each point represents a cell. One-way ANOVA performed overall (P < 0.0002) with Dunnett’s multiple comparisons test comparing all conditions to scrambled, Retro-2: * P = 0.048, siSec16A: *** P < 0.0001, siSar1A/B NS P = 0.3654. (g) Schematic model of Retro-2’s mode of action. (i) Under untreated conditions, Syn5 cycles in a Sec16A-dependent manner between Golgi and ER. (ii) GPP130 cycles between Golgi and plasma membrane. (iii) In this study, we show that GPP130 and Syn5 interact directly, and that this interaction is involved in Shiga toxin trafficking from endosomes to the Golgi. (iv) Retro-2 binds to Sec16A, reducing anterograde Syn5 trafficking, thereby leading to its relocalization to the ER. (v) Syn5 relocalization prevents its interaction with GPP130 and results in the inhibition of retrograde Shiga toxin trafficking.