V1 and V0 domains of the human H+-ATPase are linked by an interaction between the G and a subunits.
The specialized H(+)-ATPases found in the inner ear and acid-handling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown wide-spread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H(+)-ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H(+)-ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V(1) and V(0) domains in man, which is known to be required for H(+)-ATPase assembly and regulation.
Norgett
Elizabeth E
EE
Department of Medical Genetics, Department of Medicine and Division of Renal Medicine, University of Cambridge, Cambridge, UK.
Borthwick
Katherine J
KJ
Al-Lamki
Rafia S
RS
Su
Ya
Y
Smith
Annabel N
AN
Karet
Fiona E
FE
D000818:Animals; D005455:Fluorescent Antibody Technique; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D008297:Male; D051379:Mice; D009399:Nephrons / Q000201:enzymology*; D010446:Peptide Fragments / Q000276:immunology; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep / Q000276:immunology
D010446:Peptide Fragments; D019151:Peptide Library; D021122:Protein Subunits; D012333:RNA, Messenger; D006639:Histidine; D006180:Proton-Translocating ATPases