Functional characterization of a Na(+)-coupled dicarboxylate carrier protein from Staphylococcus aureus

Abstract

We have cloned and functionally characterized a Na(+)-coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na(+) symporter (DASS) family and shares significant sequence homology with the mammalian Na(+)/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na(+)-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na(+) (K(0.5) of 2.7 mM) binding before dicarboxylate (K(m) of 4.5 microM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.

FIG. 1.

Functional expression of SdcS. (A) Western blot of whole cells (optical density at 660 nm of 10; 10 μl/lane): molecular mass standards (lane 1); BL21 housing pQE-80L grown with (lane 3) and without (lane 2) IPTG; BL21 housing pQE-80L/SdcS grown with (lane 5) and without (lane 4) IPTG. Protein was detected using a monoclonal antibody directed against the N-terminal SdcS histidine tag. (B) Succinate transport activity of IPTG-induced strain BL21 housing pQE-80L (•) and pQE-80L/SdcS (○) in the presence of 100 μM succinate and 5 mM NaCl. Cells housing either pQE-80L or pQE-80L/SdcS and grown in the absence of IPTG exhibited succinate transport profiles similar to that of IPTG-induced cells carrying plasmid pQE-80L. Values shown are means ± standard errors for three independent experiments.

FIG. 2.

Kinetics of SdcS-mediated dicarboxylate transport. Initial rates of fumarate (▿), malate (▪), and succinate (•) transport were estimated as described in Materials and Methods. Data from three independent trials are shown as means ± standard errors. K_m and V_max values for SdcS under these conditions are shown in Table 1.

FIG. 3.

Effects of pH on kinetic constants for succinate transport. K_m and V_max values for SdcS are as indicated. Assays were performed as described in Materials and Methods except that assay and wash buffers used morpholineethanesulfonic acid (MES)-MOPS-Tris rather than MOPS-Tris. The concentrations of succinate used to estimate kinetic constants were 1 μM to 200 μM. Kinetic constants are reported as means ± standard errors for three independent experiments.

FIG. 4.

Cation selectivity of SdcS. Rates of cation-dependent succinate transport were measured in the presence of 5 mM salt (A), 100 mM salt (B), and 5 mM NaCl-95 mM salt (C). To maintain isotonicity, K₂SO₄ was used at one-half the noted concentrations. Transport rates were estimated after a 5-min incubation of cells with labeled substrate as described in Materials and Methods, except that assay and wash buffers contained 50 mM MOPS-Tris, pH 7, plus the noted additives. Data from three independent trials were normalized to the transport value measured in the presence of 5 mM NaCl-95 mM choline chloride (38 ± 5.5 nmol/mg protein) and are shown as means ± standard errors.

FIG. 5.

Kinetics of cation-dependent succinate transport. Initial rates of succinate transport (100 μM) in the presence of NaCl (•) and LiCl (○). Transport was estimated as described in Materials and Methods except that the assay buffer contained 50 mM MOPS-Tris, pH 7, plus the indicated concentrations of NaCl (or LiCl). Assay buffer isotonicity was maintained at 100 mM by the addition of the necessary amounts of choline chloride. Data from three independent trials were normalized to peak values (16 ± 2.9 nmol/mg protein/min for NaCl; 6.7 ± 0.49 nmol/mg protein/min for LiCl) and are shown as means ± standard errors. K_0.5 and V_max values for SdcS under these conditions are shown in Table 1.

FIG. 6.

(A) Initial rates (1 min) of succinate transport as a function of succinate at 0.5 (•), 1 (○), 2 (▾), 3.5 (▵), 5 (▪), 7.5 (□), 10 (⧫), and 20 (⋄) mM NaCl. (B) Replots of panel A as a function of Na⁺ ion concentration: 1 (⋄), 2 (⧫), 5 (□), 10 (▪), 20 (▵), 50 (▾), 100 (○), and 200 (•) μM succinate. Data from three independent trials were normalized to peak values (17 to 19 nmol/mg protein/min) and are shown as means ± standard errors. (C) A replot of apparent K_m values was used to derive true K_0.5 and K_m constants for Na⁺ and succinate assuming an ordered bireactant system in which K_B^app = {(K_A)² K_B + K_AK_B [A]}/[A]² + K_B, where A and B denote Na⁺ and succinate, respectively.