Cellular Concentrations of the Transporters DctA and DcuB and the Sensor DcuS of Escherichia coli and the Contributions of Free and Complexed DcuS to Transcriptional Regulation by DcuR.

In Escherichia coli, the catabolism of C4-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C4-dicarboxylates (like fumarate) and complexation with the C4-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuSF) is known to be constantly active even in the absence of fumarate, whereas the DcuB-DcuS and DctA-DcuS complexes require fumarate for activation. To elucidate the impact of the transporters on the functional state of DcuS and the concentrations of DcuSF and DcuB-DcuS (or DctA-DcuS), the absolute levels of DcuS, DcuB, and DctA were determined in aerobically or anaerobically grown cells by mass spectrometry. DcuS was present at a constant very low level (10 to 20 molecules DcuS/cell), whereas the levels of DcuB and DctA were higher (minimum, 200 molecules/cell) and further increased with fumarate (12.7- and 2.7-fold, respectively). Relating DcuS and DcuB contents with the functional state of DcuS allowed an estimation of the proportions of DcuS in the free (DcuSF) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuSF levels were always low (<2% of total DcuS), ruling out earlier models that show DcuSF as the major species under noninducing conditions. In the absence of fumarate, when DcuSF is responsible for basal dcuB expression, up to 8% of the maximal DcuB levels are formed. These suffice for DcuB-DcuS complex formation and basal transport activity. In the presence of fumarate (>100 µM), the DcuB-DcuS complex drives the majority of dcuB expression and is thus responsible for induction.IMPORTANCE Two-component systems (TCS) are major devices for sensing by bacteria and adaptation to environmental cues. Membrane-bound sensor kinases of TCS often use accessory proteins of unknown function. The DcuS-DcuR TCS responds to C4-dicarboxylates and requires formation of the complex of DcuS with C4-dicarboxylate transporters DctA or DcuB. Free DcuS (DcuSF) is constitutively active in autophosphorylation and was supposed to have a major role under specific conditions. Here, absolute concentrations of DcuS, DcuB, and DctA were determined under activating and nonactivating conditions by mass spectrometry. The relationship of their absolute contents to the functional state of DcuS revealed their contribution to the control of DcuS-DcuR in vivo, which was not accessible by other approaches, leading to a revision of previous models.

Expression of sodium-dependent dicarboxylate transporter 1 (NaDC1/SLC13A2) in normal and neoplastic human kidney.

Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport, and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study's purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by colocalization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of nonproximal tubule origin, oncocytoma and chromophobe carcinoma. In summary, 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1's role in human dicarboxylate metabolism.

[The change of human Na+/dicarboxylate co-transporter 1 expression in the kidney and its relationship with pathogenesis of nephrolithiasis].

OBJECTIVE: To study the change of Na+/dicarboxylate co-transporter 1 expression in the kidney and its relationship with nephrolithiasis. METHODS: 50 volunteers and 85 patients with nephrolithiasis were divided into 3 groups: control, nephrolithiasis with normal urine citrate, and nephrolithiasis with hypocitraturia. The expression of hNaDC1 mRNA in kidney was determined by RT-PCR or Northern blotting, the change of hNaDC1 protein abundance were measured by immunohistochemical staining with anti-hNaDC1 antibody among part of these patients and volunteers. The plasma and urinary biochemical parameters, such as citrate, oxalate, uric acid and calcium etc., were analyzed by routine chemical methods. RESULTS: The recurrence rate of nephrolithiasis in the group of patients with hypocitraturia was 36.1%, significantly higher than the recurrence rate of 16.3% in the group of patients with normal urine citrate (P < 0.01). hNaDC1 was expressed in the normal kidney, localized in the striated border of renal proximal tubule. However, it was expressed highly in the kidneys of patients with hypocitraturia. The ratio hNaDC1 mRNA/18sRNA in the patients with hypocitraturia was 0.65 +/- 0.21, significantly higher than that in the controls (0.36 +/- 0.11, P < 0.01). The ratio hNaDC1 mRNA/18sRNA in the patients with normal urine citrate was not significantly different from that in the controls (P > 0.05). The urine pH and urine sodium were significantly lower in the patients with hypocitraturia than in the other two groups. The levels of urine calcium and urine oxalate were significantly higher in the patients with hypocitraturia than in the controls, and were not different from those in the patients with normal urine citrate. CONCLUSION: The upregulation of hNaDC1 mRNA and protein abundance in the kidney may be an important cause of hypocitraturia, which might be related with the occurrence and recurrence of nephrolithiasis.