Identification of domains within the V-ATPase accessory subunit Ac45 involved in V-ATPase transport and Ca2+-dependent exocytosis.
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.
Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition, and Behaviour and Nijmegen Centre for Molecular Life Sciences (NCMLS), Faculty of Science, Radboud University Nijmegen, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands.
Eric J R
EJ
Jansen
Nick H M
NH
van Bakel
Nikkie F M
NF
Olde Loohuis
Theo G M
TG
Hafmans
Tim
T
Arentsen
Anthon J M
AJ
Coenen
Wim J J M
WJ
Scheenen
Gerard J M
GJ
Martens
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D010449:Peptide Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D059748:Proteolysis; D017384:Sequence Deletion; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D029867:Xenopus Proteins; C484227:atp6ap1.1 protein, Xenopus; D025262:Vacuolar Proton-Translocating ATPases; D002118:Calcium