Abstract
Mycolactone is a bacteria-derived macrolide that blocks the biogenesis of a large array of secretory and integral transmembrane proteins (TMP) through potent inhibition of the Sec61 translocon. Here, we used quantitative proteomics to delineate the direct and indirect effects of mycolactone-mediated Sec61 blockade in living cells. In T lymphocytes, dendritic cells and sensory neurons, Sec61 substrates downregulated by mycolactone were in order of incidence: secretory proteins (with a signal peptide but no transmembrane domain), TMPs with a signal peptide (Type I) and TMPs without signal peptide and a cytosolic N terminus (Type II). TMPs without a signal peptide and the opposite N terminus topology (Type III) were refractory to mycolactone inhibition. This rule applied comparably to single- and multi-pass TMPs, and extended to exogenous viral proteins. Parallel to its broad-spectrum inhibition of Sec61-mediated protein translocation, mycolactone rapidly induced cytosolic chaperones Hsp70/Hsp90. Moreover, it activated an atypical endoplasmic reticulum stress response, differing from conventional unfolded protein response by the down-regulation of Bip. In addition to refining our mechanistic understanding of Sec61 inhibition by mycolactone, our findings thus reveal that Sec61 blockade induces proteostatic stress in the cytosol and the endoplasmic reticulum.