Almost all membrane proteins are inserted or translocated across membranes by the universally conserved Sec translocon. Despite its central role, experimental access to Sec function has remained limited. Here, we present ProSecCO (Protein Secretion in Cell-free via synthetic Operons), which is a cell-free protein synthesis platform that inserts SecYEG into synthetic vesicles, enabling direct testing of Sec in real-time and high-throughput, circumventing longstanding viability constraints. Screening 300 Sec variants in a single experiment, we consolidate three decades of Sec research, while vastly expanding mutant diversity for structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, we uncover dozens of super-active translocation variants and one variant of improved insertion activity. We further leverage ProSecCO to increase membrane protein quality and nanobody export, highlighting the potential of our system for advancing applications in synthetic biology and biotechnology.
M. Meier, Scott A. Scholz, Leo von Bank et al.· Science Advances· 2 citations
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 sequences (SSs) that require additional assistance to trigger the opening of the Sec61 channel. In this study, we investigated how the hydrophobicity of SSs affects the interaction with the Sec complex at the single-molecule level using optical tweezers. We analyzed three SSs representing low, moderate, and high hydrophobicity in the h-regions. Our results show that the highly hydrophobic SS from DPAP-B pαF forms a less stable bond with the Sec complex, evidenced by shorter interaction time and reduced energy barrier to the unbound state. In contrast, SSs 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 SSs based on their hydrophobicity, offering insights into the mechanisms by which defects in SS translocation contribute to ER-associated diseases.
W. Sánchez, Luka Robeson, Hilda M. Alfaro-Valdés et al.· Biochemical and Biophysical...· 0 citations
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