Energetics of Protein Transport across Biological Membranes: A Study of the Thylakoid ΔpH-Dependent/cpTat Pathway

Among the pathways for protein translocation across biological membranes, the ΔpH-dependent/Tat system is unusual in its sole reliance upon the transmembrane pH gradient to drive protein transport. The free energy cost of protein translocation via the chloro-plast ΔpH-dependent/Tat pathway was measured by conducting in vitro transport assays with isolated thylakoids while concurrently monitoring energetic parameters. These experiments revealed a substrate-specific energetic barrier to cpTat-mediated transport as well as direct utilization of protons from the gradient, consistent with a H+/protein antiporter mechanism. The magnitude of proton flux was assayed by four independent approaches and averaged 7.9 × 104 protons released from the gradient per transported protein. This corresponds to a ΔGtransport of 6.9 × 105 kJ•mol protein translocated−1, representing the utilization of an energetic equivalent of 104 molecules of ATP. At this cost, we estimate that the ΔpH-dependent/cpTat pathway utilizes approximately 3% of the total energy output of the chloroplast.