The a-subunit of the V-type H+-ATPase interacts with phosphofructokinase-1 in humans.
V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in alpha-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in alpha-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.
Su
Ya
Y
Department of Medical Genetics, Cambridge University, United Kingdom.
Zhou
Aiwu
A
Al-Lamki
Rafia S
RS
Karet
Fiona E
FE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D006801:Humans; D007668:Kidney / Q000378:metabolism; D025221:Phosphofructokinases / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases; D025262:Vacuolar Proton-Translocating ATPases