The chloroplast Tat pathway utilizes the transmembrane electric potential as an energy source

Abstract

The thylakoid membrane, located inside the chloroplast, requires proteins transported across it for plastid biogenesis and functional photosynthetic electron transport. The chloroplast Tat translocator found on thylakoids transports proteins from the plastid stroma to the thylakoid lumen. Previous studies have shown that the chloroplast Tat pathway is independent of NTP hydrolysis as an energy source and instead depends on the thylakoid transmembrane proton gradient to power protein translocation. Because of its localization on the same membrane as the proton motive force-dependent F(0)F(1) ATPase, we believed that the chloroplast Tat pathway also made use of the thylakoid electric potential for transporting substrates. By adjusting the rate of photosynthetic proton pumping and by utilizing ionophores, we show that the chloroplast Tat pathway can also utilize the transmembrane electric potential for protein transport. Our findings indicate that the chloroplast Tat pathway is likely dependent on the total protonmotive force (PMF) as an energy source. As a protonmotive-dependent device, certain predictions can be made about structural features expected to be found in the Tat translocon, specifically, the presence of a proton well, a device in the membrane that converts electrical potential into chemical potential.

FIGURE 1

Valinomycin decreases the rate of cpTat transport at low values of ΔpH. (A) The import data of four ionophore treatments replicated nine times are shown, as well as triplicates of 10% substrate loading standards used for quantification purposes interspersed with a nonradioactive molecular weight marker (empty lanes). The average percentage transport of each treatment as well as the standard errors and t-tests are presented graphically (B). The two pairs of columns labeled Control and Nigericin show the percentage transport of iOE17 Tat substrate under high-light conditions in the absence and presence of 300 nM of nigericin. Light shaded bars are untreated reactions, and solid bars are reactions that contain 1 μM valinomycin.

FIGURE 2

ECS of carotenoids indicates the electric potential in thylakoids. The ECS values presented are the deflection of carotenoid absorbance from a dark baseline to a steady-state level achieved during a 1-min actinic illumination period. (A) Effect of valinomycin when the ΔpH was lowered using increasing nigericin concentrations. (B) Effect of valinomycin when the ΔpH was lowered using decreasing actinic light intensities. In both panels the two paired columns represent the ECS in the absence (solid bars) or presence (light shaded bars) of 1 μM valinomycin. The standard error is based on three to four replicates of fourfold signal-averaged ECS measurements. (C–F) Selected paired traces of ECS measurements of which the steady state magnitude was used in creating the above graphs. Of the paired traces, in all cases, the steady-state electric potential–indicating ECS signal that developed without a rapid spike of electrochromism at the onset of illumination was the treatment containing 1 μM valinomycin. (C, D) The two paired ECS traces were each performed at an actinic intensity of 20 μE s⁻¹·m⁻², but (C) without nigericin and (D) with 1.5 nM nigericin. (E, F) The two paired ECS traces consist of experiments performed with actinic light intensities of 20 and 10 μE s⁻¹·m⁻², respectively.

FIGURE 3

Protein transport as a function of ΔpH attenuated with nigericin (A) or actinic light intensity (B). Protein transport is plotted against ΔpH determined by 9-AA fluorescence quenching in the absence (solid circles) or presence of 1 μM valinomycin (light shaded diamonds). Insets represent models of the components of the PMF in a thylakoid during a ΔpH titration with either nigericin (A) or actinic intensity (B). Solid lines represent the total PMF, which is the sum of the ΔpH and the Δψ; dashed lines represent the Δψ; dot-dashed line represents the ΔpH; solid and light shaded lines represent titrations in the presence or absence of valinomycin, respectively.

FIGURE 4

Protein transport during a 1-min reaction is affected by Δψ when the ΔpH is titrated with light. The attenuated thylakoid ΔpH was set by nigericin addition (A) or by low actinic light intensity (B). Averages and standard errors of six replicate treatments are shown without (solid circles) and with (light shaded squares) 1 μM valinomycin, respectively, in otherwise identical conditions and (B) at a lower light intensity to reset the ΔpH in the presence of 1 μM valinomycin (light shaded triangle) to that without valinomycin.