OEP40, a Regulated Glucose-permeable β-Barrel Solute Channel in the Chloroplast Outer Envelope Membrane

Abstract

Chloroplasts and mitochondria are unique endosymbiotic cellular organelles surrounded by two membranes. Essential metabolic networking between these compartments and their hosting cells requires the exchange of a large number of biochemical pathway intermediates in a directed and coordinated fashion across their inner and outer envelope membranes. Here, we describe the identification and functional characterization of a highly specific, regulated solute channel in the outer envelope of chloroplasts, named OEP40. Loss of OEP40 function in Arabidopsis thaliana results in early flowering under cold temperature. The reconstituted recombinant OEP40 protein forms a high conductance β-barrel ion channel with subconductant states in planar lipid bilayers. The OEP40 channel is slightly cation-selective PK+/PCl- ≈ 4:1 and rectifying (i⃗/i⃖ ≅ 2) with a slope conductance of Ḡmax ≅ 690 picosiemens. The OEP40 channel has a restriction zone diameter of ≅1.4 nm and is permeable for glucose, glucose 1-phosphate and glucose 6-phosphate, but not for maltose. Moreover, channel properties are regulated by trehalose 6-phosphate, which cannot permeate. Altogether, our results indicate that OEP40 is a "glucose-gate" in the outer envelope membrane of chloroplasts, facilitating selective metabolite exchange between chloroplasts and the surrounding cell.

Keywords: beta-barrel protein; chloroplast; glucose transport; ion channel; membrane transport; metabolite transport; outer membrane; solute channel.

FIGURE 1.

OEP40 forms a β-barrel in the chloroplast OE membrane. A, immunoblot analysis of Ps-OEP40 in chloroplast subfractions. Equal protein amounts (5 μg) of pea chloroplast OE, IE, stroma (str), and thylakoids (thy) were separated by SDS-PAGE and subjected to immunoblot analysis using antibodies directed against recombinant Ps-OEP40. For detection with antisera against marker proteins LSU (str), LHCP (thy), PIC1 (IE), and OEP16.1 (OE), different protein amounts (i.e. 1.0, 0.6, 10.0, and 1.0 μg, respectively) were loaded in each lane. Numbers indicate molecular mass of proteins in kDa. B, three-dimensional structure of a 10-stranded At-OEP40 β-barrel generated by TMBpro. For clarity, the mainly unstructured C terminus starting from His²³⁴ (compare supplemental Figs. S1 and S3) is not shown.

FIGURE 2.

Single channel recording from planar bilayers containing reconstituted OEP40. A, single channel recordings in symmetrical buffer conditions (250/250 mm KCl) from bilayers containing a single active OEP40 channel in response to an applied voltage gate of the indicated voltage amplitude (V_m). B, mean variance histogram of the current recording at V_m = 100 mV (top) and V_m = −100 mV (bottom). C, mean variance plot of the current recording at V_m = 100 mV (top) and V_m = −100 mV (bottom). D, current-voltage ramp (−100 to 100 mV, sweep-rate 15 mV/s) of a single open OEP40 channel in symmetrical buffer (see A). E, current-voltage ramp (−80 to 80 mV, sweep-rate 15 mV/s) of a single open OEP40 channel in asymmetrical buffer (250/20 mm KCl). F, voltage-dependent open probability of OEP40 single channels (n = 3).

FIGURE 3.

Effect of glucose on the single channel activity of reconstituted OEP40. A, single channel recordings in symmetrical buffer (250/250 mm KCl) from a bilayer containing a single active OEP40 channel in response to an applied voltage gate of 60 s duration with the indicated voltage amplitude (V_m). B and C, single channel recordings in symmetrical buffer from the same bilayer as in A but in the presence of 6 mm glucose (cis/trans). D, voltage dependence of the fast (τ_fast) and slow dwell-times (τ_slow) of glucose-induced transitions between the fully open and closed state of the OEP40 channel (6 mm glucose, cis/trans). E, voltage dependence of the frequency of τ_fast and τ_slow of glucose (6 mm, cis/trans)-induced transitions between the fully open and closed state of the OEP40 channel (n = 3). F, current-voltage ramp (−80 to 80 mV, sweep-rate 15 mV/s) of an OEP40 channel in symmetrical buffer conditions (see A) with the addition of 6 mm glucose only in cis. For a control without glucose, see Figs. 2D and 4C (upper panel).

FIGURE 4.

Effect of Glc-1-P on the single channel activity of reconstituted OEP40. A, current-voltage ramp (−70 to +70 mV, sweep-rate 15 mV/s) of a single open OEP40 channel in the absence (control) and presence of 6 mm Glc-1-P in the cis compartment, which corresponds to the low conductance site of the OEP40 channel (symmetrical 20/20 mm KCl buffer conditions). B, same as A except symmetrical 100/100 mm KCl buffer conditions. C, same as A except symmetrical 250/250 mm KCl buffer conditions.

FIGURE 5.

Effect of Tre-6-P on single channel activity of reconstituted OEP40. A, current recording from a bilayer containing a single active OEP40 channel in response to an applied voltage gate of V_m = −60 mV in the absence (top) and presence of 1 mm Tre-6-P (cis/trans). Symmetrical buffer conditions (250/250 mm KCl). B, voltage-dependent open probabilities (n = 3) of the OEP40 channel in the absence (black square) and presence of 1 mm Tre-6-P (red circle) (cis/trans). C, apparent conductance G_max obtained from the maximal current amplitudes (I_max) at the given voltages in the absence (black square) and presence of 1 mm Tre-6-P (red circle) (n = 3). D, current-voltage ramp (−80 to 80 mV sweep-rate 15 mV/s) of a single open OEP40 channel in symmetrical buffer (20/20 mm KCl) in the presence of 1 mm Tre-6-P in the cis compartment. E, current-voltage ramps (−80 to 80 mV sweep-rate 15 mV/s) of a single open OEP40 channel in asymmetrical buffer (250/20 mm KCl) in the absence (upper panel) and presence of 1 mm Tre-6-P in cis (lower panel). F, same as E except the asymmetric 20/100 (cis/trans) KCl buffer conditions.

FIGURE 6.

Arabidopsis mutants with reduced OEP40 protein display an early flowering phenotype a low temperature conditions. A and B, representative individuals of 64- and 85-day-old-plants of Col-0 wild-type and oep40-1 and oep40-3 homozygous mutant lines grown at 10 °C. C and D, DTB (left) and TLN (right) were recorded in plant lines described in A, B. Plants were grown at 10 °C in C and at 21 °C (day) and 16 °C (night) in D. Bars depict mean numbers ± S.D. of n = 27 individual plants for each line. Asterisks indicate significant differences in DTB and TLN in oep40 mutants compared with Col-0 (Student's t test, p < 0.05). Right graphs, black bars represent rosette leaf (RLN) and white bars cauline leaf numbers (CLN). Please note that first flowers of oep40-1 and oep40-3 appeared about 8–9 days earlier than for Col-0 wild type.

FIGURE 7.

Floral transition occurs earlier in oep40 mutant lines compared with wild type. Emergence of floral primordia was visualized by RNA in situ hybridization using a specific LFY probe on longitudinal sections through apices of Col-0 wild type as well as homozygous oep40-1 and oep40-3 mutant lines. The plants were grown under low temperature conditions (10 °C) and harvested every 2nd day over a period of 3 weeks. Representative images of 34–54-day-old plants are shown as indicated. Asterisks indicate the meristem summit.