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Summary: The emergence of lipid membranes and embedded proteins was essential for the evolution of cells. Translocon complexes mediate cotranslational recruitment and membrane insertion of nascent proteins, but they already contain membrane-integral proteins. Therefore, a simpler mechanism must exist, enabling spontaneous membrane integration while preventing aggregation of unchaperoned protein in the aqueous phase. Here, we used giant unilamellar vesicles encapsulating minimal translation components to systematically interrogate the requirements for insertion of the model protein proteorhodopsin (PR) – a structurally ubiquitous membrane protein. We show that the N-terminal hydrophobic domain of PR is both necessary and sufficient for cotranslational recruitment of ribosomes to the membrane and subsequent membrane insertion of PR. Insertion of N-terminally truncated PR was restored by artificially attaching ribosomes to the membrane. Our findings offer a self-sufficient protein-inherent mechanism as a possible explanation for effective membrane protein biogenesis in a “pretranslocon” era, and they offer new opportunities for generating artificial cells.

The emergence of lipid membranes and embedded proteins was essential for the evolution of cells. Translocon complexes mediate co-translational recruitment and membrane insertion of nascent proteins, but they already contain membrane-integral proteins. Therefore, a simpler mechanism must exist, enabling spontaneous membrane integration while preventing aggregation of un-chaperoned protein in aqueous phase. Here, we used giant unilamellar vesicles encapsulating a minimal protein synthesis kit to systematically interrogate the requirements for membrane insertion of proteorhodopsin (PR) – a structurally ubiquitous membrane protein. We show that the N-terminal hydrophobic domain of PR is both necessary and sufficient for co-translational recruitment of ribosomes to the membrane and subsequent membrane insertion of PR. Insertion of N-terminally truncated PR was restored by artificially attaching ribosomes to the membrane. Our findings offer a self-sufficient protein-inherent mechanism as a possible explanation for effective membrane protein biogenesis in a ‘pre-translocon’ era, and they offer new opportunities for generating artificial cells.