The conserved membrane-proximal domain of Sbh1/ Sec61β guides signal peptides into the Sec61 channel

Protein secretion begins with protein translocation through the universally conserved Sec61 channel in the endoplasmic reticulum (ER) membrane. The function of its β-subunit, called Sbh1 in yeast, remains poorly understood. Sbh1/Sec61β has a cytosolic domain consisting of two parts: the N-terminal approximately 40 amino acids are intrinsically unstructured, followed by a conserved membrane proximal (CMP) domain. Sbh1 is anchored to the Sec61 channel by a single C-terminal transmembrane domain. Phosphorylation of the Sbh1/Sec61β intrinsically unstructured domain allows regulated import of specific proteins into the ER. We show here that S3/T5 phosphorylation of Sbh1 results in a conformational change in the Sbh1 intrinsically unstructured N-terminus. We also address the function of the Sbh1/Sec61β CMP domain. We identify a potential interaction site in the Sbh1 CMP domain with the Sec61 N-terminal amphipathic helix using molecular dynamics (MD) modelling. Peptide panning revealed that the Sbh1 CMP domain contains a binding site for Sbh1-dependent signal peptides. Using site-directed mutagenesis, we show that the CMP region controls import of Sbh1-dependent translocation substrates into the ER. We conclude that the Sbh1 CMP domain functions as a gatekeeper that recognizes and guides specific substrates towards the Sec61 channel for insertion.