Ferrous ion transport across chloroplast inner envelope membranes

Abstract

The initial rate of Fe(2+) movement across the inner envelope membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Fe(2+)-sensitive fluorophore, Phen Green SK. The rate of Fe(2+) transport was rapid, coming to equilibrium within 3s. The maximal rate and concentration dependence of Fe(2+) transport in predominantly right-side-out vesicles were nearly equivalent to those measured in largely inside-out vesicles. Fe(2+) transport was stimulated by an inwardly directed electrochemical proton gradient across right-side-out vesicles, an effect that was diminished by the addition of valinomycin in the presence of K(+). Fe(2+) transport was inhibited by Zn(2+), in a competitive manner, as well as by Cu(2+) and Mn(2+). These results indicate that inward-directed Fe(2+) transport across the chloroplast inner envelope occurs by a potential-stimulated uniport mechanism.

Figure 1

Ferrous iron quenching of PGSK fluorescence in asolectin and chloroplast inner envelope membrane vesicles. Asolectin and chloroplast inner envelope membrane vesicles were loaded with PGSK using the extrusion method described in “Materials and Methods” (pH = 8.0). Vesicles were rapidly mixed with 5 μm Fe²⁺ in external buffer (pH 7.0) in a stopped-flow apparatus and fluorescence emission was monitored at 520 nm with excitation at 506 nm. A, Asolectin vesicles. B, Asolectin vesicles pre-incubated 10 min with 20 mm pyrithione. C, Chloroplast inner envelope membrane vesicles.

Figure 2

Ferrous iron quenching of PGSK fluorescence in chloroplast inner envelope membrane vesicles. Chloroplast inner envelope membrane vesicles were loaded with PGSK using the extrusion method described in “Materials and Methods” (pH = 8.0). Vesicles were mixed with varying amounts of Fe²⁺ in external buffer (pH 7.0) as indicated. Fluorescence emission was monitored at 520 nm after excitation at 506 nm.

Figure 3

Stern-Volmer plot of PGSK fluorescence quenching by cations. The fluorescence emission, monitored at 520 nm with excitation at 506 nm of a solution of 50 μm PGSK in buffer A, was measured in the presence of varying concentrations of Fe²⁺, Zn²⁺, or Mn²⁺.

Figure 4

Intravesicular concentration of ferrous iron in chloroplast inner envelope membrane vesicles loaded with PGSK. Data from Figure 2 were used to determine the intravesicular Fe²⁺ concentration using the calibration procedure outlined in “Materials in Methods.”

Figure 5

Initial rate of Fe²⁺ transport in extruded and freeze-/thaw-prepared chloroplast inner envelope membrane vesicles. Inner envelope membranes were prepared by extrusion to produce vesicles largely right side out in orientation. Inner envelope membranes were also prepared by a freeze/thaw technique to produce membranes of largely inside-out orientation. Internal vesicle pH was 8.0, whereas external pH was 7.0. Intravesicular Fe²⁺ concentration was determined as described in “Materials and Methods” at different concentrations of added Fe²⁺. Initial rates were determined from the equation describing a single exponential increase.

Figure 6

Effect of external pH on the initial rate of Fe²⁺ transport across chloroplast inner envelope membrane vesicles. PGSK-loaded inner envelope vesicles (pH = 8.0 inside) were mixed with external buffer at different pH values plus 5 μm Fe²⁺. PGSK fluorescence quenching was determined over 3 s after mixing and, using the calibration procedure described in “Materials and Methods,” the initial rates of transport were determined.

Figure 7

pH and potential gradient effects on Fe²⁺ transport across chloroplast inner envelope membrane vesicles. A, Extruded inner envelope vesicles at pH 8.0 were mixed with external buffer at pH 8.0 containing 5 μm Fe²⁺. B, Extruded inner envelope vesicles at pH 8.0 were mixed with external buffer at pH 7.0 containing 5 μm Fe²⁺. C, Conditions were the same as B with vesicles pre-incubated with 2 nm valinomycin on ice for 30 min. D, Membrane potential was imposed across the vesicle membranes by mixing vesicles prepared in 100 mm KCl, 10 mm K-HEPES, and 100 mm Suc with an equal volume of the same buffer in which the 100 mm KCl was replaced with 100 mm choline chloride. The instantaneous equilibration of K⁺ in the presence of valinomycin resulted in a negatively charged vesicle interior. Data shown are the mean ± sd (n = 9).

Figure 8

Effect of added cations on Fe²⁺ transport across chloroplast inner envelope membrane vesicles. Vesicles contained buffer at pH 8.0 inside and were mixed with external buffer at pH 7.0. Control rate of Fe²⁺ transport into PGSK-loaded vesicles was established using 5 μm Fe²⁺. Addition of all other cations was also at 5 μm. Intravesicular Fe²⁺ concentration was determined as described in “Materials and Methods” at different concentrations of added Fe²⁺. Initial rates were determined from the equation describing a single exponential increase. Data shown are the mean ±sd (n = 3).

Figure 9

Lineweaver-Burk plot of Zn²⁺ inhibition of Fe²⁺ transport. Inner envelope membranes were prepared by extrusion. Intravesicular Fe²⁺ concentration was determined as described in “Materials and Methods” at different concentrations of Fe²⁺ in the absence (●) and presence (▪) of 5 μm Zn²⁺.