Breaking bonds: A single-molecule study of rupture forces and hydrophobicity in signal peptide-sec complex interaction

The Sec61 complex plays a critical role in protein translocation into the endoplasmic reticulum (ER), enabling the passage of soluble proteins into the lumen and the lateral insertion of membrane proteins. The Sec complex, comprising Sec61, Sec63, Sec62, and other auxiliary proteins, is especially important for the translocation of precursor proteins with inefficient signal peptides (SPs) that require additional assistance to trigger the opening of the Sec61 channel. In this study, we investigated how the hydrophobicity of SPs affects the interaction with the Sec complex by optimizing and implementing a single-molecule force spectroscopy approach with optical tweezers. We analyzed three SPs representing low, moderate, and high hydrophobicity in the h-regions. Our results show that the highly hydrophobic SP forms a less stable bound with the Sec complex, evidenced by shorter interaction time and reduced energy barrier to the unbound state. In contrast, SPs with lower hydrophobicity establish more stable interactions, characterized by extended interaction times, and elevated energy barriers to reach the unbound state. These findings suggest that the Sec complex may selectively recognize and interact with SPs based on their hydrophobicity, offering insights into the mechanisms by which defects in SP translocation contribute to ER-associated diseases.