Molecular mechanisms of cutis laxa- and distal renal tubular acidosis-causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4

J Biol Chem. 2018 Feb 23;293(8):2787-2800. doi: 10.1074/jbc.M117.818872. Epub 2018 Jan 8.


The a subunit is the largest of 15 different subunits that make up the vacuolar H+-ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1-a4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 (a2) and ATP6V0A4 (a4) subunits and their mutants, a2P405L (causing cutis laxa), and a4R449H and a4G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N-Glycosylation was assessed using endoglycosidases, revealing that a2P405L, a4R449H, and a4G820R were fully N-glycosylated. Cycloheximide (CHX) chase assays revealed that a2P405L and a4R449H were unstable relative to wildtype. a4R449H was degraded predominantly in the proteasomal pathway, whereas a2P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a4R449H and defective Golgi trafficking of a2P405L Co-immunoprecipitation studies revealed an increase in association of a4R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4G820R, where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa.

Keywords: 3D modeling; ATPase; ER-associated degradation; N-glycosylation; membrane protein; protein degradation; proton pump; trafficking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acidosis, Renal Tubular / genetics*
  • Acidosis, Renal Tubular / metabolism
  • Acidosis, Renal Tubular / pathology
  • Amino Acid Substitution
  • Cell Membrane / enzymology
  • Cell Membrane / metabolism
  • Cell Membrane / pathology
  • Cutis Laxa / genetics*
  • Cutis Laxa / metabolism
  • Cutis Laxa / pathology
  • Endoplasmic Reticulum / enzymology
  • Endoplasmic Reticulum / metabolism
  • Endoplasmic Reticulum / pathology
  • Enzyme Stability
  • Glycosylation
  • Golgi Apparatus / enzymology
  • Golgi Apparatus / metabolism
  • Golgi Apparatus / pathology
  • HEK293 Cells
  • Humans
  • Kidney / enzymology
  • Kidney / metabolism
  • Kidney / pathology
  • Models, Molecular*
  • Mutation, Missense*
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Interaction Domains and Motifs
  • Protein Multimerization
  • Protein Processing, Post-Translational*
  • Protein Transport
  • Proteolysis
  • Proton-Translocating ATPases / chemistry
  • Proton-Translocating ATPases / genetics*
  • Proton-Translocating ATPases / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Vacuolar Proton-Translocating ATPases / chemistry
  • Vacuolar Proton-Translocating ATPases / genetics*
  • Vacuolar Proton-Translocating ATPases / metabolism


  • ATP6V0A2 protein, human
  • ATP6V1B1 protein, human
  • Recombinant Fusion Proteins
  • Proteasome Endopeptidase Complex
  • ATP6V0A4 protein, human
  • VMA21 protein, human
  • Vacuolar Proton-Translocating ATPases
  • Proton-Translocating ATPases

Associated data

  • PDB/5I1M