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In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na+/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.

Summary Intestinal transport of 32P-labelled inorganic phosphate was measured in everted gut sacs, and scraped enterocytes prepared from segments along the length of small intestine, from duodenum to the terminal ileum of hypophosphatemic male ( Hyp /Y) mice and normal (+/Y) littermates. Uptake of phosphate by purified brush-border membrane vesicles prepared from the small intestine of both genotypes was also measured. We found no deficiency of phosphate transport in the Hyp small intestine. In vitro transport of phosphate by sacs and enterocytes is greatest in the distal intestinal segments of mouse.

Because arachidonic acid and its metabolites are reported to be intracellular messengers of various exogenous stimuli, we studied whether arachidonic acid influences phosphate transport by cultured mouse renal epithelial cells. Arachidonic acid, at 10 −7 –10 −4 M, inhibited phosphate transport without influencing cyclic adenosine 3′:5′-monophosphate production. Nordihydroguaiaretic acid and indomethacin, inhibitors of arachidonic acid metabolism, did not cancel the arachidonic acid-induced inhibition of phosphate transport. Furthermore, unsaturated fatty acids other than arachidonic acid also inhibited phosphate transport and their inhibitory effect increased as the number of double bond increased. These data demonstrate that arachidonic acid inhibits the phosphate transport by the cultured renal epithelial cells, probably not via conversion to its metabolites.

Summary Submitocondrial particles prepared from bovine heart mitochondria in the presence of CaCl 2 on MnCl 2 take up inorganic phosphate. The reaction is sensitive to mersalyl and to hydrophobic N-benzylmaleimide but is insensitive to N-ethylmaleimide. These findings indicate that the phosphate transport system is oriented asymmetrically.

Summary Effects of parathyroid hormone (PTH), low phospohate environment, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) on the phosphate reabsorption by the renal tubular cells from mutant hemizygous hypophosphatemic (Hyp/Y) mice and their littermates (+/Y) were studied using a phosphate accumulation system which had been developed recently. This system mimics phosphate transport at the renal tubules. When cultured in a normal phosphate medium, the characteristics of the phosphate accumulation by Hyp cells was almost identical with that by normal cells; a PTH-induced inhibition and a TPA-induced stimulation of phosphate accumulation. However, when preincubated in a low phosphate medium, the accumulation of phosphate by normal cells increased significantly, while that by Hyp cells did not. These results indicate that the adaptation to the low phosphate environment is defective in Hyp cells and it may be one of the cause of renal phosphate leakage in the Hyp mouse.

N-ethylmaleimide inhibits the mitochondrial phosphate carrier. Mitochondria were titrated with N-[ 3 H]ethylmaleimide, dissolved in dodecylsulfate-mercaptoethanol, and their proteins separated on dodecylsulfate-polyacrylamide gels. While the phosphate transport is essentially insensitive to low concentrations of N-ethylmaleimide, the six primary N-ethylmaleimide reactive inner membrane proteins are labeled in direct proportion to the amount of inhibitor added. The reaction of N-[ 3 H]ethylmaleimide with proteins I and III is independent of the preincubation of the mitochondria with p-mercuribenzoic acid, a membrane impermeable inhibitor of the transport. Comparing the alkylation of proteins II, IV, V and VI with the inhibition of phosphate transport, it is found that only proteins IV (45,000 daltons) and V (32,000 daltons) are maximally labeled at the same N-[ 3 H]ethylmaleimide concentration that maximally inhibits the transport.

Tribenzylphosphate (TBP), a specific inhibitor of the high affinity system for Pi transport in yeast mitochondria, inhibits the active Pi transport measured by the energy-linked swelling. The depence of the rate of oligomycin sensitive ATP synthesis as a function of the external Pi concentration shows two kinetic systems. The high affinity system, corresponds to the range of the external Pi concentration which stimulates the respiratory rate. TBP inhibits both this system and the state 4 → state 3 transition.

The stoicheiometry of respiratory driven proton translocation associated with the oxidation of endogenous substrates has been measured in an organic phosphate auxotrophic mutant of Escherichia coli . The results obtained indicate that movements of inorganic phosphate do not result in an experimental underestimation of the observed H+/site ratio in E. coli , as has previously been suggested for mitochondria.