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A novel concept in eukaryotic signal transduction is the use of nutrient transporters and closely related proteins as nutrient sensors. The action mechanism of these 'transceptors' is unclear. The Pho84 phosphate transceptor in yeast transports phosphate and mediates rapid phosphate activation of the protein kinase A (PKA) pathway during growth induction. We have now identified several phosphate-containing compounds that act as nontransported signaling agonists of Pho84. This indicates that signaling does not require complete transport of the substrate. For the nontransported agonist glycerol-3-phosphate (Gly3P), we show that it is transported by two other carriers, Git1 and Pho91, without triggering signaling. Gly3P is a competitive inhibitor of transport through Pho84, indicating direct interaction with its phosphate-binding site. We also identified phosphonoacetic acid as a competitive inhibitor of transport without agonist function for signaling. This indicates that binding of a compound into the phosphate-binding site of Pho84 is not enough to trigger signaling. Apparently, signaling requires a specific conformational change that may be part of, but does not require, the complete transport cycle. Using Substituted Cysteine Accessibility Method (SCAM) we identified [Phe.sup.160] in TMD lV and [Val.sup.392] in TMD VIII as residues exposed with their side chain into the phosphate-binding site of Pho84. Inhibition of both transport and signaling by covalent modification of [Pho84.sup.F160C] or [Pho84.sup.V392C] showed that the same binding site is used for transport of phosphate and for signaling with both phosphate and Gly3P. Our results provide to the best of our knowledge the first insight into the molecular mechanism of a phosphate transceptor. nutrient sensing | protein kinase A | growth induction | Saccharomyces cerevisiae www.pnas.org/cgi/doi/10.1073/pnas.0906546107