N-linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H + -ATPase (V-ATPase) biosynthesis

J Cell Biochem. 2018 Jan;119(1):861-875. doi: 10.1002/jcb.26250. Epub 2017 Aug 4.

Abstract

The a subunit of the V0 membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope-tagged human a1-a3 subunits, with or without mutations that convert Asn to Gln at putative N-glycosylation sites. Expression and N-glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N-glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild-type a subunits after enzymatic deglycosylation. Cycloheximide-chase experiments showed that unglycosylated subunits were turned over at a higher rate than N-glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co-immunoprecipitation studies showed that they were unable to associate with the V-ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N-glycosylation is crucial in all four human V-ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V-ATPase complexes.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Asparagine / genetics
  • Binding Sites
  • Endoplasmic Reticulum / metabolism
  • Glutamine / genetics
  • Glycosylation / drug effects
  • HEK293 Cells
  • Humans
  • Mutation
  • Protein Binding
  • Protein Biosynthesis
  • Protein Stability
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Vacuolar Proton-Translocating ATPases / chemistry*
  • Vacuolar Proton-Translocating ATPases / genetics*
  • Vacuolar Proton-Translocating ATPases / metabolism*

Substances

  • Protein Subunits
  • Glutamine
  • Asparagine
  • Vacuolar Proton-Translocating ATPases