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
Membraneless organelles play essential roles in many cellular processes. In various photosynthetic organisms, they are a crucial part of CO2/carbon-concentrating mechanisms (CCMs) that increase photosynthetic productivity. One example is the pyrenoid in Chlamydomonas reinhardtii, a liquid-phase-separated organelle that localizes and improves CO2 fixation via the intrinsically disordered protein essential pyrenoid component 1 (EPYC1CR). Modern-day pyrenoids are complex structures with an elaborate cellular architecture and dozens of components, raising the question of how they could have developed from simpler condensates. Here we develop a bottom-up approach to study the function of EPYC1s and explore their sequence–function space across phylogenetic diversity and evolution. We demonstrate that extant and ancestral EPYC1 sequences induce phase separation of Rubisco into synthetic pyrenoids with functional CCMs. Surprisingly, these CCMs are mainly based on enhanced carboxylation rates (rather than increased specificity), offering new insights into the construction, function and evolution of natural and synthetic pyrenoids. This study shows that the structural protein essential pyrenoid component 1 (EPYC1) and the enzyme Rubisco are sufficient to assemble into pyrenoid-like structures that are functionally active and show a basic carbon-concentrating activity.
A. Küffner, B. Pommerenke, L. Kley et al.· Nature Plants· 0 citations
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