Transport of dicarboxylates in Saccharomyces cerevisiae

Transport of dicarboxylates plays an important role in cell metabolism. In particular, they are intermediates of the citrate cycle. Transport of succinate across the mitochondrial membrane provides correlation between metabolism in peroxysomes and in mitochondria. In recent years, much attention has been given to transport of dicarboxylates across plasma membranes of animal, plant and bacterial cells. However, yeast cell transporters have not been studied systematically. The results of our research group experiments for the period 2001-2010 years are summarized in this minireview. Our previous experiments showed specific features of changes in levels of endogenous respiration substrates in Saccharomyces cerevisiae cells at low temperature. The rate of endogenous respiration of cells in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30°C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h, indicating the physiological significance of L-malate in regulation of the S. cerevisiae cell respiration. A nonconventional approach to the measurement of succinate transport across plasmalemma is proposed. It is based on the conditions in which the succinate oxidation rate is limited by transport across plasmalemma. Transport of succinate into S. cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the Km value of the mitochondrial “succinate oxidase” was fivefold less than that of the cellular “succinate oxidase”. O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dickson plots indicates that the rate of succinate oxidation is limited by its transport across plasmalemma. The plasma membrane of S. cerevisiae was found to have a carrier catalyzing the transport of dicarboxylates (succinate, L-malate and malonate). This approach allowed for the reproducible determination of Km for the dicarboxylate transporter (7.3 ± 2.1 mM) within a half-hour period. We found that plasmalemmal dicarboxylate transporter is also involved in citrate influx and is modulated by pH and cations. Succinate and citrate transport into yeast cells was studied by measuring substrate oxidation rates in the presence and in the absence of effective impermeable oxidation inhibitors. Linearity of the Dickson plot for 2-undecyl malonate suggests that this inhibitor blocked the rate-limiting step upon oxidation of both substrates. This approach allowed fast (within 30– 40 min) measurement of kinetic parameters of the transporter in individual samples. Succinate and citrate transport was insensitive to the protonophore FCCP, being activated by Na ions and competitively inhibited by 2-undecyl malonate and K ions. Values of Ki for 2-undecyl malonate were similar for both substrates. These data suggest that citrate and succinate influx is mediated by a common plasma membrane transporter. This is not typical for fungi. Topography of the active site of the S. cerevisiae plasmalemmal dicarboxylate transporter was studied using lipophilic derivatives of its substrates (2alkylmalonates and O-acyl-L-malates). Probing of the active site of this transporter has revealed a large lipophilic area stretching between the 0.72 to 2.5 nm from the substrate-binding site. Itaconate inhibited the transport fivefold more effectively than L-malate. This suggests the existence of a hydrophobic region immediately near the dicarboxylate-binding site (to 0.72 nm). Fumarate but not maleate competitively inhibited succinate transport into the cells. It is suggested that the plasmalemmal transporter binds the substrate in the trans-conformation. The prospects of the proposed approach for scanning lipophilic profiles of channels of different transporters are discussed.