UCSF

Cotransins are a class of cyclic-heptadepsi-peptides that potently inhibits translocation of a subset of proteins across the membrane of the endoplasmic reticulum (ER). Sensitivity or resistance to cotransins depends on the identity of a protein's N-terminal ER-targeting sequence. However, the precise mechanism of action of cotransins is unknown. Motivated by cotransins' unprecedented ability to block an essential step in the biogenesis of several therapeutically relevant secretory and membrane proteins, we sought to clarify their precise mechanism of action.

We discovered that cotransins directly target a highly conserved protein-conducting channel in the ER membrane known as Sec61. Sec61 mediates the translocation of secretory proteins across the ER membrane and the insertion of most integral membrane proteins into the ER membrane. Despite directly binding to a channel utilized by all proteins entering the ER, cotransins block passage of only a select few. These findings lead us to hypothesize that cotransins could be used as chemical probes into the underlying molecular mechanism of Sec61 function.

To test this possibility, we determined how cotransins block Sec61-mediated insertion of a model protein into the ER membrane. We found that cotransin-bound Sec61 impedes passage of a hydrophobic transmembrane domain (TMD) from the pore of Sec61 into the surrounding lipid bilayer, thereby permitting analysis of a previously uncharacterized pre-integration intermediate. Site-specific crosslinking studies of this intermediate revealed an α-helical TMD docked near the cytosolic face of the lateral gate in Sec61. Progression through the cotransin-stabilized stage was strongly influenced by biophysical properties of the TMD, such as α-helical propensity and hydrophobicity. Cotransins therefore reveal that direct interactions between the TMD and the lateral gate of Sec61 precede TMD transfer into the membrane.

Taken together, these findings identify Sec61 as a possible therapeutic target and provide a mechanistic framework in which to judge the therapeutic potential of cotransins. Future studies will seek to define the entire subset of human proteins whose expression is inhibited by cotransins and the efficacy of cotransins in different models of human disease.