Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association

Int J Mol Sci. 2023 Mar 2;24(5):4867. doi: 10.3390/ijms24054867.

Abstract

Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.

Keywords: PI(4,5)P2; V-ATPase a4 isoforms; V-ATPases; dRTA; phosphoinositides; protein–lipid interaction.

MeSH terms

  • Animals
  • Binding Sites
  • Cell Membrane / metabolism
  • HEK293 Cells
  • Humans
  • Mammals / metabolism
  • Phosphatidylinositols / metabolism
  • Protein Isoforms / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Vacuolar Proton-Translocating ATPases* / metabolism

Substances

  • Vacuolar Proton-Translocating ATPases
  • Protein Isoforms
  • Phosphatidylinositols