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

Abstract

AbstractProtein 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.