Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum

Production and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-associated degradation (ERAD) eeyarestatin 1 (ES_I) is a potent inhibitor of protein translocation. We have characterised this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ES_I targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ES_I acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ES_I, and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.