V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.
The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH-dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.
Jansen
Eric J R
EJ
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, Nijmegen, The Netherlands.
Hafmans
Theo G M
TG
Martens
Gerard J M
GJ
D000143:Acids / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D052717:Melanotrophs / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D008970:Molecular Weight; D051859:Neuroendocrine Secretory Protein 7B2 / Q000378:metabolism; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis*; D045664:Proprotein Convertase 1 / Q000378:metabolism; D045706:Proprotein Convertase 2 / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000521:alpha-MSH / Q000378:metabolism
D000143:Acids; D004791:Enzyme Inhibitors; D018942:Macrolides; D051859:Neuroendocrine Secretory Protein 7B2; D021122:Protein Subunits; D029867:Xenopus Proteins; C484227:atp6ap1.1 protein, Xenopus; D049452:Green Fluorescent Proteins; D000521:alpha-MSH; D011333:Pro-Opiomelanocortin; C040929:bafilomycin A1; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D025262:Vacuolar Proton-Translocating ATPases