The conserved membrane-proximal domain of Sbh1/ Sec61β guides signal peptides into the Sec61 channel
In eukaryotes, protein secretion begins with protein translocation through the universally conserved Sec61 channel into the endoplasmic reticulum (ER). Its {beta}-subunit, Sbh1 in yeast, enhances ER import of proteins with specific suboptimal signal peptides by an unknown mechanism. The Sbh1 cytosolic N-terminus consists of an intrinsically disordered, non-conserved region (IDR) that has never been visualized in active channel structures, but is close to the translocating polypeptide in the cytosolic channel vestibule. The Sbh1/Sec61{beta} N-terminal IDR is followed by structured 15 amino acids and its C-terminal transmembrane helix, both of which are conserved. We show here that the proline and adjacent conserved residues at the Sbh1 cytosolic/transmembrane domain interface form a hinge that positions the Sbh1 cytosolic domain across the channel vestibule and orients it with respect to the lateral gate. This orientation is critical for Sbh1-dependent protein insertion into the channel. Sbh1-dependence of Sec61 channel insertion is a function of the signal peptide of the respective secretory protein. By chemical crosslinking of purified cytosolic domains of Sbh1 and its paralog Sbh2 to synthetic signal peptides derived from their respective client proteins, we show that the cytosolic domains of Sbh1 and Sbh2 contain specific signal peptide binding sites. The position of the crosslinked residues suggests that signal peptide binding is mediated by the IDRs. We conclude that Sec61{beta} homologs directly recognize signal peptides of their substrates and guide them into the Sec61 channel; they thus control entry of specific proteins into the secretory pathway.