Thylakoid targeting of Tat passenger proteins shows no ΔpH dependence in vivo

The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane ΔpH. In this paper, we assess the ΔpH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the ΔpH in vivo. Using experimental conditions in which the trans-thylakoid ΔpH was almost zero, we found no evidence for a ΔpH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a ΔpH under physiological conditions, but becomes ΔpH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors.