The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.
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Data and code availability
The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier project accession no. PXD015642. Imaris and Matlab scripts for EEA1 quantification are available on request. All other data supporting the findings of this study are available within the paper and its supplementary information files.
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We thank R. Rodriguez (cell imaging of Retro-2.1 and target identification), G. Boncompain (RUSH), C. Viaris De Lesegno (PLA), R. Onclercq-Delic and S. Bombard (intoxication assays), C. Brewee and B. Sancerne (recombinant protein production and cytotoxicity assays), D. Buisson (purification of 15) and E. Chirkin (synthesis of new batches of chemicals) for help with the indicated experiments. We thank V. Sabatet from the Laboratoire de Spectrométrie de Masse Protéomique for myProMS assistance. We acknowledge support from grants from the Agence Nationale pour la Recherche (ANR-11-BSV2-0018 and ANR-14-CE16-0004-03 to L.J., J.B., J.-C.C. and D.G. and ANR-19-CE13-0001-01 to L.J.), the Human Frontier Science Program (RGP0029-2014 to L.J.), the European Research Council (advanced grant no. 340485 to L.J.), the Swedish Research Council (K2015-99X-22877-01-6 to L.J., J.-C.C. and D.G.), the Joint Ministerial Program of R&D against CBRNE Risks (D.G., J.B., J.-C.C. and L.J.), the CEA (D.G., J.B. and J.-C.C.), the Île de France Region DIM Malinf initiative (grant no. 140101 to D.G., J.B. and L.J.), the Région Île-de-France (D.L.) and the Fondation pour la Recherche Médicale (D.L.). The Gillet and Cintrat teams are members of LabEx LERMIT (ANR-10-LABX-33) and the Johannes team is a member of Labex CelTisPhyBio (11-LBX-0038) and Idex Paris Sciences et Lettres (ANR-10-IDEX-0001–02 PSL). We also acknowledge the Cell and Tissue Imaging (PICT-IBiSA) and Nikon Imaging Centre, Institut Curie, a member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).
The authors declare no competing interests.
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Supplementary Tables 1–2, Figs. 1–7 and Supplementary Note.
Proteomics quantification results GFP-Sec16A vehicle versus EGFP.
Proteomics quantification results GFP-Sec16A Retro-2 versus GFP-Sec16A vehicle.
Proteomics quantification results GFP-STX5 versus EGFP.
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Forrester, A., Rathjen, S.J., Daniela Garcia-Castillo, M. et al. Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2. Nat Chem Biol 16, 327–336 (2020). https://doi.org/10.1038/s41589-020-0474-4
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