The importance of sorting proteins and wall materials to their destination is critical for plant growth and development, though the machinery orchestrating membrane trafficking is poorly understood. Transporters that alter the environment across endomembrane compartments are thought to be important players. Using Escherichia coli and yeast, we previously showed that several Arabidopsis Cation/H(+) eXchanger (AtCHX) members were K(+) transporters with a role in pH homeostasis, though their subcellular location and biological roles in plants are unclear. Co-expression of markers with CHX16, CHX17, CHX18, or CHX19 tagged with a fluorescent protein indicated these transporters associated with plasma membrane (PM) and post-Golgi compartments. Under its native promoter, AtCHX17(1-820)-GFP localized to prevacuolar compartment (PVC) and to PM in roots. Brefeldin A diminished AtCHX17-GFP fluorescence at PM, whereas wortmannin caused formation of GFP-labeled ring-like structures, suggesting AtCHX17 trafficked among PVC, vacuole and PM. AtCHX17(1-472) lacking its carboxylic tail did not associate with PVC or PM in plant cells. Single chx17 mutant or higher-order mutants showed normal root growth and vegetative development. However, quadruple (chx16chx17chx18chx19) mutants were reduced in frequency and produced 50%-70% fewer seeds, indicating overlapping roles of several AtCHX17-related transporters in reproduction and/or seed development. Together, our results suggest that successful reproduction and seed development depend on the ability to regulate cation and pH homeostasis by AtCHX17-like transporters on membranes that traffic in the endocytic and/or secretory pathways.

Endomembrane system cation/H+ exchangers affect male gametophyte and sperm function through membrane trafficking that is likely to remodel the plasma membrane and cell walls.

Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na(+)/H(+) antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. These results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H(+) exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.

No abstract