Purification and functional characterization of the vacuolar malate transporter tDT from .

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of overexpressors, CO feeding and quantification of C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, overexpressors exhibited malate and citrate levels opposite to knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.