Ion transport across intracellular membranes is essential for cellular homeostasis, yet organellar transport mechanisms remain difficult to resolve in native cells. Here, we establish a cell-free membrane system to interrogate full-length intracellular transporters under defined chemical conditions. Using cell-free protein synthesis, human Golgi transporter TMEM165 and its yeast homolog GDT1 were synthesized in membranes and analyzed by fluorescence and electrochemical measurements. The platform controlled protein identity, ion composition, and transmembrane pH gradients. TMEM165 transport was regulated by pH gradients, consistent with proton-coupled transport shaped by the electrostatic environment of the transport pathway. Disruption of a conserved acidic residue decreased pH sensitivity, broadened ion permeability, and altered steady-state transport behavior, indicating impaired proton coupling and selectivity. These findings identify conserved acidic residues as key regulators of TMEM165 function. More broadly, this work establishes a strategy for reconstructing organellar membrane proteins, resolving disease mechanisms, and engineering transport function in defined synthetic systems.
Selivanovitch, E., Ostwalt, A., Byra, A., Chao, Z., Gaiffe, A., van Raemdonck, G., Askowitz, O., Morsomme, P., Carten, J., Daniel, S.
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