Measurement of the Energetics of Protein Transport Across the Chloroplast Thylakoid Membrane

Protein transport across cellular membranes represents an unknown, possibly significant drain on the total energy pool. Many protein transport systems utilize a mixture of energetic inputs, with contributions from both NTP hydrolysis and transmembrane electrochemical gradients. Both of these parameters will have to be measured before we can know the cost to the cell of its considerable protein transport activities. We describe here methods to evaluate the magnitude of the ΔpH across the thylakoid membrane, which serves as the driving force for protein transport on the cpTat pathway, and to determine how much energy is drained therefrom per protein translocated. The methods derive from spectroscopic techniques, well known in the field of thylakoid energetics, to monitor the light-dependent ΔpH across the membrane and the rate of proton flux through the thylakoid lumen, combined with those to measure the rate of protein transport across the thylakoid membrane.