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Adenosine triphosphate (ATP), the cellular energy currency, is essential for life. The ability to provide a constant supply of ATP is therefore crucial for the construction of artificial cells in synthetic biology. Here, we describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorganic phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD + , and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to co-reconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addition of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidation, ubiquinone cycling, oxygen reduction, proton pumping, and ATP synthesis. As a proof-of-principle, we demonstrate that our nanovesicles are capable of using an NAD + -linked substrate to drive cell-free protein expression. Our nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.