Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis

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.

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left–right asymmetry in zebrafish

“…V-ATPase accessory proteins are known to regulate V-ATPase activity (Jansen et al, 2010a; Jansen et al, 2008; Jansen et al, 2012), but these proteins could also have V-ATPase-independent functions. We took two approaches to test whether KV phenotypes in Atp6ap1b depleted embryos are linked to altered V-ATPase function.…”

Regulation of synaptic vesicle acidification at the neuronal synapse

“…Of note, proteomics studies on purified SVs have also revealed the presence of ATP6AP1 (ATPase H + Transporting Accessory Protein 1, also known as Ac45) and ATP6AP2 (ATPase H + Transporting Accessory Protein 2, also known as the (pro)renin receptor/PRR) . Those two brain‐enriched trans‐membrane proteins are accessory components of the vATPase with some additional cellular functions—for example, ATP6AP1 is involved in the vATPase transport and exocytosis, and ATP6AP2 is reported to be essential for biogenesis of active vATPases . ATP6AP2 is also ideally posed to link vATPase to cell signaling, for example, the renin‐angiotensin system for regulating blood pressure and electrolyte balance, as well as Wnt signaling during embryo development .…”

“…Of note, knock‐outs of the essential vATPase subunits are lethal, as well as of accessory subunit ATP6AP1, making the studies of these proteins difficult. If not lethal, a genetic perturbation of the vATPase subunits and accessory proteins ATP6AP1 and ATP6AP2 lead to inherited disorders …”

“…If not lethal, a genetic perturbation of the vATPase subunits and accessory proteins ATP6AP1 and ATP6AP2 lead to inherited disorders. 20,21,61…”

Ac45 silencing mediated by AAV‐sh‐Ac45‐RNAi prevents both bone loss and inflammation caused by periodontitis

“…Ac45 (Atp6ap1), an accessory subunit of the V-ATPase complex, is a type I transmembrane protein associated with the V-ATPase membrane domain (V0) (Yang et al 2012). The domains located in the N-and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca2 + -dependent-regulated exocytosis (Jansen et al 2012). Recently, overexpression of an Ac45 cytoplasmic terminus deletion mutant in RAW264.7 cells resulted in a dramatic reduction in osteoclast-mediated bone resorption and alterations in the binding proximity of Ac45 with the V-ATPase V0 domain subunits a3, c″ and d (Yang et al 2012).…”