Energetic studies of thecpTat protein transport pathway

The cpTat protein transport pathway is uniquely suited to in vitro energetic studies because of its location on the energy-transducing thylakoid membrane. The cpTat pathway is reliant solely on the thylakoid ApH as an energy source: There is no involvement of NTP hydrolysis, and previous studies have shown that transport is independent of the membrane potential (DeltaPsi) component of the thylakoid protonmotive force. Importantly, the proton chemical potential of the thylakoid---on which the cpTat pathway relies to power transport---decays rapidly in the dark.; This dissertation demonstrates the reliance of the cpTat pathway on DeltaPsi to power cpTat transport. This was accomplished by careful measurements of the electric potential in thylakoids using actinic light intensity attenuation and ionophore titration. From subsequent transport assays under identical conditions, the reliance of the cpTat pathway on the electric potential component of the protonmotive force has now been demonstrated. Reliance on both the DeltapH and DeltaPsi implies that the cpTat pathway is dependent on the total protonmotive force, and thus suggests structural constraints that would be present on a functional cpTat transporter.; This work also demonstrates the ability of the cpTat pathway to transport substrates in the dark, seemingly without any energy. It was established that dark transport genuinely occurred on the cpTat pathway and that thylakoid protonmotive force was not arising from artifactual means. The use of indicating dyes to measure the thylakoid potential in the dark demonstrated no DeltapH or DeltaPsi present in the thylakoids a short time after actinic illumination ended. These findings support the hypothesis that the cpTat pathway is reliant on a pool of protons out of equilibrium from the bulk aqueous proton pool.