Abstract
The vacuolar H+-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position −561/−547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H+-ATPase complex at these locations.
Highlights
Vacuolar-type H+-ATPases (v-ATPases) consist of two major subunits: a membrane anchoring V0 domain and a catalytic V1 domain
While there is a generic structure of v-ATPase proton pumps derived from assemblage of ubiquitously expressed subunits e.g. A1 and E2 many cell types present a particular holoenzyme composition based on tissue or cell specific expression of subunit isoforms e.g. B1 and a4 that are expressed in epithelia of inner ear, kidney and epidiymis [5]
While the different subunits as expected are expressed at the apical aspect of epithelial FORE cells Foxi1 staining is localized to the nuclei (Fig. 1 B, E, H, K)
Summary
Vacuolar-type H+-ATPases (v-ATPases) consist of two major subunits: a membrane anchoring V0 domain and a catalytic V1 domain. These multimeric enzymes are mediators of both intraand extracellular vectorial proton transport in all eukaryotic cells. This type of proton pump is enriched for in the plasma membrane of cells specialized in proton secretion such as FORE cells of the endolymphatic sac and duct [1,2], intercalated cells of the collecting duct in the kidney [3] and narrow and clear cells of epididymis [4]. In spite of a growing body of information regarding structure – function relationships of v-ATPase [9], intracellular targeting and recycling [10] as well as how the coupling ratio between ATP hydrolysis and proton transport is modulated [11] little is known regarding upstream regulators of subunit gene expression
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