Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties.

Structure-function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter.