Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae

Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na+/P-i cotransporter with twelve putative transmembrane domains, the YBR296e ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PNO89, was cloned and a set of deletion mutants was constructed. We then studied their P-i uptake activity under different conditions. We show here that a transport activity displayed by PNO89 strains under alkaline conditions and in the presence of Na+ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na+ was omitted, this activity decreased significantly, reaching values close to those exhibited by the Delta pho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PNO89 gene is under the control of the PNO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of Pi limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na+/P+ cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent K-m of 0.5 mu M and a pH optimum of 9.5, and is highly specific for Na+; activation of transport is maximal at a Na+ concentration of 25 mM.