Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent KM values were determined with 10.12μM, 4.85μM and 19.95μM, respectively for NAT3 and 1.74μM, 2.44μM and 29.83μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton-nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1-7 and TM8-14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure-function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.

Edited by Dennis R. Voelker Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PLtransfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuliindependent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.

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