Energetic cost of protein import across the envelope membranes of chloroplasts

Abstract

Chloroplasts are the organelles of green plants in which light energy is transduced into chemical energy, forming ATP and reduced carbon compounds upon which all life depends. The expenditure of this energy is one of the central issues of cellular metabolism. Chloroplasts contain ~3,000 proteins, among which less than 100 are typically encoded in the plastid genome. The rest are encoded in the nuclear genome, synthesized in the cytosol, and posttranslationally imported into the organelle in an energy-dependent process. We report here a measurement of the amount of ATP hydrolyzed to import a protein across the chloroplast envelope membranes--only the second complete accounting of the cost in Gibbs free energy of protein transport to be undertaken. Using two different precursors prepared by three distinct techniques, we show that the import of a precursor protein into chloroplasts is accompanied by the hydrolysis of ~650 ATP molecules. This translates to a ΔG(protein) (transport) of some 27,300 kJ/mol protein imported. We estimate that protein import across the plastid envelope membranes consumes ~0.6% of the total light-saturated energy output of the organelle.

Fig. 1.

Inhibition by tentoxin of background ATPase activity in chloroplasts. Chloroplasts were incubated at room temperature in the dark for 10 min in IB containing tentoxin as indicated. ATP mixed with [γ-³²P]-ATP was then added to final concentration of 1 mM. Samples were withdrawn at 0 and 30 min after adding ATP. Reactions were stopped by adding an ice-cold acidic charcoal solution. After separating Pi from ATP and other nucleotides using charcoal (Materials and Methods), released [³²P] was quantified by scintillation counting. Each point on the graph is the mean of at least three replicates; error bars represent SD.

Fig. 2.

Effects of inhibitors of different chloroplast biochemical pathways on background ATPase activity. (A) Chloroplasts were resuspended in IB containing (or not) tentoxin (5 μM), DCMU (10 μM), and where indicated, a mixture of the inhibitors bispyribac-sodium, chloramphenicol, glyphosate, halosulfuron-methyl, penoxsulam, spectinomycin, imazethapyr, clomazone, malathion, and rifampicin, was added, bringing each to the specified final concentration. Chloroplasts were pretreated at room temperature in the dark for 15 min, and then ATP was added to 1 mM (cold ATP mixed with [³²P]-ATP). Samples were removed at 0 and 60 min and ATPase activities were determined. (B) Chloroplasts in IB supplemented with 1 mM DTT were incubated at room temperature for 30, 60, and 120 min in the absence or presence of inhibitors. A total of 1 mM ATP ([³²P] and cold) was added to start the ATPase reaction at room temperature in the dark. Samples were withdrawn at 0 and 30 min and ATPase activities were determined. Control, no inhibitors; inhibitor cocktail, each inhibitor mentioned at the indicated concentrations; TTX/DCMU, 5 μM tentoxin and 10 μM DCMU; TTX/DCMU + inhibitors, 5 μM tentoxin, 10 μM DCMU, each inhibitor mentioned above at 100 μM. Each point on the graphs is a mean of at least three replicates; error bars represent SD.

Fig. 3.

Effects of ATP concentration on background ATPase activity and protein import. Chloroplasts were incubated in the presence of 10 μM DCMU and 5 μM tentoxin at room temperature in the dark for 15 min. Buffer (A) or bacterially expressed tp22-GFP (B), 1 mM DTT and the indicated concentration of ATP were added to start the reactions. Samples were removed at 0, 15, 30, 45, and 60 min. (A) Background ATPase activity. Each point on the graph is the mean of at least three replicates; error bars represent SD. (B) Import assay.

Fig. 4.

Protein import assayed by immunoblotting. Bacterially expressed tp22-GFP was incubated with isolated chloroplasts in the presence of 3 mM ATP in the dark at room temperature for 0 or 60 min, followed by thermolysin (TL, +) or mock protease treatment (−). Samples containing 4 μg Chl were separated by SDS/PAGE followed by immunoblotting against GFP antibody (Upper) or Coomassie staining (Lower). The left three lanes show the signal intensities from the indicated amounts of tp22-GFP loaded onto the gel. LHCP, light harvesting chlorophyll-binding protein; m, mature form of tp22-GFP; p, tp22-GFP; SSU, small subunit of RuBisCO.