Transport in C₄ mesophyll chloroplasts. Characterization of the pyruvate carrier

1. Evidence is presented for high rates of carrier-mediated uptake of pyruvate into the stroma of intact mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis, but not the chloroplasts of the C₃ plant Spinacea oleracea. Uptake of pyruvate in the dark with the C₄ mesophyll chloroplasts was followed using two techniques: uptake of [¹⁴C]pyruvate as determined by silicon oil centrifugal filtration and uptake as indicated by absorbance changes at 535 nm (shrinkage/swelling) after addition of 0.1 M pyruvate salts. 2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [¹⁴C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium. 3. Uptake of pyruvic acid (or a pyruvate ^-/OH^- antiport) is ruled unlikely since [¹⁴C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations. 4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [¹⁴C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (-80 mV at low ionic strength). 5. Chloride and inorganic phosphate were potent inhibitors of [¹⁴C]pyruvate uptake. Of the sulfhydryl reagents tested, N-ethylmaleimide was not inhibitory while mersalyl completely blocked [¹⁴C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C. 6. Phenylpyruvate, α-ketoisovalerate, α-ketoisocaproate, α-cyano-4-hydroxycinnamic acid and α-cyanocinnamic acid inhibited [¹⁴C]pyruvate but not [¹⁴C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light.