Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Significance Aside from its undisputed role in the import of newly synthesized proteins into the endoplasmic reticulum (ER), the Sec61 translocon was proposed to ensure the reverse transport of misfolded proteins to the cytosol. Based on this model, Sec61 was also proposed to be the channel exporting internalized antigens from endosomes to the cytosol, for degradation and cross-presentation. Establishing Sec61’s contribution to these connected trafficking pathways has nevertheless proven difficult, due to a technical incapacity to blunt its activity acutely. Here, we took advantage of a recently identified Sec61 blocker to determine whether or not Sec61 can mediate retrograde protein transport. Both ER-to-cytosol and endosome-to-cytosol protein export were intact in mycolactone-treated cells, which argues against Sec61 operating as a retrotranslocon. Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61’s contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8+ T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.