Identification of domains within the V-ATPase accessory subunit Ac45 involved in V-ATPase transport and Ca2+-dependent exocytosis

J Biol Chem. 2012 Aug 10;287(33):27537-46. doi: 10.1074/jbc.M112.356105. Epub 2012 Jun 26.

Abstract

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium / metabolism*
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / metabolism*
  • Exocytosis / physiology*
  • Peptide Mapping / methods
  • Protein Structure, Tertiary
  • Protein Transport / physiology
  • Proteolysis*
  • Sequence Deletion
  • Vacuolar Proton-Translocating ATPases / genetics
  • Vacuolar Proton-Translocating ATPases / metabolism*
  • Xenopus Proteins / genetics
  • Xenopus Proteins / metabolism*
  • Xenopus laevis

Substances

  • Xenopus Proteins
  • atp6ap1.1 protein, Xenopus
  • Vacuolar Proton-Translocating ATPases
  • Calcium