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The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

Evidence is provided that amyloplasts from pea roots contain a translocator which transports, in a counter exchange mode, phosphate, glucose 6-phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate. The translocator has a low affinity for 2-phosphoglycerate and glucose 1-phosphate. Metabolite transport was measured by silicone oil filtering centrifugation either directly by uptake of radioactive labelled compounds or indirectly by back exchange.

The phosphate/pyrophosphate translocase protein (T2) of the hepatic microsomal glucose-6-phosphatase system was identified and then purified using antibodies raised against the rat mitochondrial phosphate/hydroxyl ion antiport protein. The T2 protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having type 1c glycogen storage disease.

In a cultured, renal epithelial cell line, OK, parathyroid hormone (PTH) reduces Na/phosphate cotransport and upon removal of the hormone the activity is regained. Cycloheximide, an inhibitor of protein synthesis, did not interfere with the PTH action, but prevented the reduced phosphate transport from regaining its original activity. Drugs, such as colchicine, that disrupt the microtubular network, lessened both the action of PTH and prevented the recovery of activity. The results are consistent with the irreversible inactivation of a plasma membrane protein necessary for full activity of the Na/phosphate cotransport.

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy-terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae. Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84-GFP chimera consistent with the behavior of the wild-type Pho84 protein. The GFP-tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e. the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation. By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo.

A maleimide spin label strongly inhibits the phosphate/H+ symporter of rat liver mitochondria. While inducing half-maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.

The phosphate transport through the inner mitochondrial membrane has been found to be inhibited by thiol reagents [ 1,2]. We have reported previously that this inhibition was different with regard to two kinds of SH-reagent: maleimides, and Ellman’s reagent were partial or total inhibitors according to the experimental conditions, however, mersalyl completely and consistently blocked phosphate transport, no matter the conditions applied [3]. From this observation and the analysis of the reactivity and permeability of some maleimides, it was proposed that Pi-carrier could be regarded as a mobile or reorientating carrier [3,4]. We found earlier that phos phate was able to protect its carrier from weak SHreagent, but not from mersalyl [4,5]. This effect seems due mainly to an orientation of SH-groups of the carrier towards the inner side, according to the observation that internal phosphate is more effective than the external one [4]. It is shown in this communication that mersalyl removes phosphate when added after a phosphate accumulation is achieved (see also ref. [6]). This observation is discussed in relation to the hypothesis of the mobile or reorientating carrier.

Isolated chick kidney proximal tubule cells have been used in a study of the mechanism by which PTH inhibits Na+-dependent Pi transport in the kidney. Treatment with PTH inhibits Pi uptake by the cells by 13% and stimulates cyclic AMP production by 77%. Forskolin, a potent activator of adenyl cyclase, brought about an 11-fold stimulation of cyclic AMP production by the cells, but in contrast to PTH, the drug had no effect on Na+-dependent Pi uptake. These results provide evidence that PTH action on phosphate transport is not mediated by cyclic AMP.