The Na(+)-P(i) cotransporter NaPi-IIb (SLC34A2) has been described to be involved in mouse small intestinal absorption of P(i) and to be regulated by a number of hormones and metabolic factors. However, a possible segmental expression of NaPi-llb in small intestine has not been addressed so far. Here, we describe that the NaPi-IIb cotransporter is highly abundant in the ileum of mouse small intestine, whereas it is almost absent in the duodenum and in the jejunum. Na(+)-P(i) cotransport studies with isolated brush border membranes confirmed that NaPi-IIb cotransport is highest in the ileum. Upregulation by a low-phosphate diet of NaPi-IIb and NaPi-IIb cotransport was observed both in the jejunum and the ileum. Furthermore, evidence is provided that a low-phosphate diet provokes an increase of the NaPi-IIb mRNA abundance along the entire small intestine. These data suggest that in mouse small intestine, phosphate is absorbed transcellulary by an Na(+)-dependent pathway in the ileum, whereas in the duodenum and jejunum, this pathway is of minimal importance. Furthermore, we conclude that along the entire mouse small intestine, low-phosphate diet affects transcription and/or the stability of NaPi-IIb mRNA.

rg Biber. Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor-and 1␣OHase-deficient mice.

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPiIIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes. Molecular & Cellular Proteomics 7: 1362-1377, 2008.

During metabolic acidosis, P i serves as an important buffer to remove protons from the body. Pi is released from bone together with carbonate buffering protons in blood. In addition, in the kidney, the fractional excretion of phosphate is increased allowing for the excretion of more acid equivalents in urine. The role of intestinal P i absorption in providing Pi to buffer protons and compensating for loss from bone during metabolic acidosis has not been clarified yet. Inducing metabolic acidosis (NH 4Cl in drinking water) for 2 or 7 days in mice increased urinary fractional P i excretion twofold, whereas serum Pi levels were not altered. Na ϩ -dependent Pi transport in the small intestine, however, was stimulated from 1.89 Ϯ 3.22 to 40.72 Ϯ 11.98 pmol/mg protein (2 days of NH 4Cl) in brush-border membrane vesicles prepared from total small intestine. Similarly, the protein abundance of the Na ϩ -dependent phosphate cotransporter NaPi-IIb in the brush-border membrane was increased 5.3-fold, whereas mRNA levels remained stable. According to immunohistochemistry and real-time PCR NaPi-IIb expression was found to be mainly confined to the ileum in the small intestine, and this distribution was not altered during metabolic acidosis. These results suggest that the stimulation of intestinal P i absorption during metabolic acidosis may contribute to the buffering of acid equivalents by providing phosphate and may also help to prevent excessive liberation of phosphate from bone. phosphate SEVERAL MECHANISMS CONTRIBUTE to the buffering and elimination of excessive protons and acid equivalents during metabolic acidosis. Besides respiration, increased release of buffer substances from bone and stimulated reabsorption of bicarbonate and increased excretion of protons by the kidneys are the major mechanisms to restore acid-base balance (11, 16). Excretion of protons by the kidney requires so called titratable acids, i.e., ammonia, citrate, and phosphate, buffering protons in urine in the collecting duct and thus increasing the maximal acid excretion rate (16). The renal excretion of ammonia and phosphate is highly increased during metabolic acidosis, whereas excretion of citrate is decreased (3). Ammonia (NH 3 ) is synthetized from glutamine metabolism in the kidney proximal tubule in response to metabolic acidosis (27). In addition, it is thought that inhibition of renal reabsorption of phosphate contributes mainly to increased phosphate excretion (1). In a rat model for metabolic acidosis, a decrease in mRNA and protein of the major Na ϩ -P i cotransporter NaPi-IIa as well as in Na ϩ -dependent P i uptake into brush border membrane (BBM) vesicles (BBMV) has been found (1). Systemic P i levels are only slightly decreased during metabolic acidosis, an effect that has been attributed to the acid-stimulated release of P i from bone together with Ca 2ϩ and carbonate (23). Indeed, the release of phosphate from bone during short-and long-term metabolic acidosis in in vivo and in in vitro models has been extensively documented a...

Abstract. The type IIa Na + /P i cotransporter (NaPiIIa) plays a key role in the reabsorption of inorganic phosphate (P i ) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTOP.