Energetics of Protein Transport Across Biological Membranes. a Study of the Thylakoid DeltapH-dependent/cpTat Pathway

Among the pathways for protein translocation across biological membranes, the DeltapH-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 DeltapH-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 x 10(4) protons released from the gradient per transported protein. This corresponds to a DeltaG transport of 6.9 x 10(5) kJ.mol protein translocated(-1), representing the utilization of an energetic equivalent of 10(4) molecules of ATP. At this cost, we estimate that the DeltapH-dependent/cpTat pathway utilizes approximately 3% of the total energy output of the chloroplast.