Identification and biochemical characterization of the SLC9A7 interactome

Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na+, K+)/H+ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.