PROJECT SUMMARY The regulation of systemic acid-base homeostasis by renal intercalated cells (IC) involves complex sensing, signaling and proton-ATPase (V-ATPase) activation pathways that remain poorly understood. New information generated during the prior funding period has shed some light on this knowledge gap by identifying new families of proteins that associate strongly with the V-ATPase to regulate its function. Based on data from our proteomic interactome screen, coupled with additional insight from RNAseq and functional assays that show their selective expression in ICs, we will focus on two of the highest scoring interacting proteins, NCOA7 (nuclear receptor co-activator 7) and DMXL1 (Drosophila melanogaster X-like gene 1). Our preliminary data clearly implicate these proteins in acidification processes in the kidney, and V-ATPase activity in renal epithelial cells. NCOA7 null mice have a distal RTA and markedly decreased V-ATPase subunit expression in ICs, while DMXL1 knockdown in cells causes deficient vesicle acidification to the same degree as knockdown of bone-fide V-ATPase subunits. Aim 1 is designed to uncover the role of NCOA7 in V-ATPase subunit expression, focusing on their translational, transcriptional and degradation regulation in WT and knockout mice. We will examine the effect of acid challenge on V-ATPase and NCOA7 functional expression and interaction. Aim 2 proposes the novel hypothesis that V-ATPase accumulation on the plasma membrane of ICs in response to systemic acid/base cues requires, counter-intuitively, a partial disassembly of the large, sterically hindering V-ATPase holoenzymes that coat transport vesicles. This is necessary to allow vesicle exocytosis and fusion, and increased pump delivery to the cell surface during the homeostatic response to systemic acidosis. We propose that DMXL1, like its yeast homolog Rav1p, is necessary for regulating pump assembly by interacting with specific V-ATPase subunits. This coordination between (dis)assembly and trafficking is required for V-ATPase membrane accumulation and increasing proton secretion by ICs. Finally, in both Aims 1 and 2, we will use a comprehensive array of protein interaction assays to identify the V-ATPase subunits involved in these associations, and to define the binding sites on NCOA7 and DMXL1 that bind to specific V-ATPase subunits. Our studies make use of a series of integrated cell and molecular techniques in conjunction with genetically modified mouse studies, isolated ICs and renal cell cultures in vitro, and interaction domain studies using purified proteins and specific subdomains. Our ultimate goal is to uncover novel causes of acidosis conditions in humans, and to inform future drug development efforts designed to modulate V- ATPase proton pumping activity, not only in acidosis (resulting from too little V-ATPase activity) but also in other pathologies such as cancer, which is often associated with inappropriately increased V-ATPase activity.