Nonmitochondrial ATP/ADP Transporters Accept Phosphate as Third Substrate

Chlamydiales and Rickettsiales as metabolically impaired, intracellular pathogenic bacteria essentially rely on “energy parasitism” by the help of nucleotide transporters (NTTs). Also in plant plastids NTT-type carriers catalyze ATP/ADP exchange to fuel metabolic processes. The uptake of ATP^4-, followed by energy consumption and the release of ADP^3-, would lead to a metabolically disadvantageous accumulation of negative charges in form of inorganic phosphate (P_i) in the bacterium or organelle if no interacting P_i export system exists. We identified that P_i is a third substrate of several NTT-type ATP/ADP transporters. During adenine nucleotide hetero-exchange, P_i is cotransported with ADP in a one-to-one stoichiometry. Additionally, P_i can be transported in exchange with solely P_i. This P_i homo-exchange depends on the presence of ADP and provides a first indication for only one binding center involved in import and export. Furthermore, analyses of mutant proteins revealed that P_i interacts with the same amino acid residue as the γ-phosphate of ATP. Import of ATP in exchange with ADP plus P_i is obviously an efficient way to couple energy provision with the export of the two metabolic products (ADP plus P_i) and to maintain cellular phosphate homeostasis in intracellular living “energy parasites” and plant plastids. The additional P_i transport capacity of NTT-type ATP/ADP transporters makes the existence of an interacting P_i exporter dispensable and might explain why a corresponding protein so far has not been identified.Most organisms possess the capacity to resynthesize the fundamental energy currency ATP by fusion of ADP and P_i. Generally, in eukaryotes the major part of energy is produced in specialized organelles, the mitochondria. Mitochondrial ADP/ATP carriers (AACs)² mediate the export of newly synthesized ATP in strict counter-exchange with cytosolic ADP and therefore provide energy to the cellular metabolism (1). Plants additionally generate high amounts of ATP during photosynthesis in chloroplasts. However, under conditions of limiting or missing photosynthetic activity, plant plastids depend on external energy supply (2–4). Specific nucleotide transporters (NTTs) located in the inner plastid envelope membrane mediate the required energy import (5). These transporters structurally, functionally, and phylogenetically differ from mitochondrial AACs. They catalyze the import of cytosolic ATP in exchange with stromal ADP, are monomers consisting of 12 predicted transmembrane helices, and are related to the functionally heterogeneous group of bacterial NTTs (5).Although most prokaryotic organisms are able to regenerate ATP and therefore are considered as energetically self-sustaining, the obligate intracellular living bacterial orders Chlamydiales and Rickettsiales are impaired in energy and nucleotide synthesis or even completely lost the corresponding pathways (6–8). Therefore, these bacteria, which comprise important human pathogens (9, 10), essentially rely on nucleotide and energy import. Bacterial NTTs catalyze the required import of a broad range of nucleotides and NAD or facilitate the counter-exchange of ATP and ADP (5, 11–15). The latter process has been termed “energy parasitism” and obviously is of high importance for the survival of rickettsial and chlamydial cells (5, 16–18).Although import measurements on intact Escherichia coli cells expressing the corresponding proteins allowed characterization of many bacterial and plastidial NTTs (12–15, 19–24), a very important physiological question is still not clarified. The uptake of ATP^4- in exchange with ADP^3- in absence of a concerted P_i export would result in a charge difference and a phosphate imbalance in the bacterial cell. In mitochondria, phosphate carriers metabolically cooperate with AACs because they provide P_i for ATP synthesis (25). Similarly, it was assumed that NTT-type ATP/ADP transporters cooperate with phosphate exporters to guarantee phosphate homeostasis in the bacterium or plastid. However, a P_i exporter interacting with ATP/ADP transporters is not known in “energy parasites” or plant plastids. Bacterial and plant phosphate transport systems rather facilitate P_i import or the counter-exchange of P_i and phosphorylated compounds and therefore do not allow net P_i export (26–29). Furthermore, the newly identified plastidial (proton-driven) phosphate transporters are not preferentially expressed under conditions or in tissues that require ATP provision to the plastid (30, 31).Recently, we succeeded in the purification of the first recombinant NTT from Protochlamydia amoebophila (PamNTT1), a parachlamydial endosymbiont of the protist Acantamoeba (32). The functional reconstitution of the highly pure PamNTT1 into artificial lipid vesicles for the first time allowed the biochemical characterization of a representative nonmitochondrial ATP/ADP transporter unaffected by the complex metabolic situation of the bacterial cell. We demonstrated that in contrast to mitochondrial AACs, PamNTT1 catalyzes a membrane potential independent, electroneutral adenine nucleotide hetero-exchange (32, 33). The latter could argue for a cotransport of a counterion compensating for the electrogenic ATP^4-/ADP^3- exchange.Here, we investigated possible ions accompanying ATP or ADP transport. Interestingly, we uncovered that PamNTT1 and also rickettsial and plastidial ATP/ADP transporters accept an additional important substrate, which is P_i. We performed a comprehensive characterization of the P_i transport and gained new insights into the transport properties of ATP/ADP transporters.