title: Mechanisms of pH regulation in the regulated secretory pathway.
abstract: A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pH(ER) = 7.4 +/- 0.2, mean +/- S.D.) to Golgi (pH(G) = 6.2 +/- 0.4) to mature secretory granules (MSGs) (pH(MSG) = 5.5 +/- 0.4). Golgi and MSGs required active H(+) v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H(+) leak rates across each membrane. However, neither steady-state pH(MSG) nor rates of passive H(+) leak were affected by Cl(-)-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pH(MSG). Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl(-) conductances. Measurements of H(+) leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical model to determine the H(+) permeability (P(H+)) of each organelle membrane. We found that P(H+) decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases in P(H+) and successive increases in the active H(+) pump density.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Wu
authors.forename: M
authors.initials: M
authors.lastname: Grabe
authors.forename: S
authors.initials: S
authors.lastname: Adams
authors.forename: R Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: H P
authors.initials: HP
authors.lastname: Moore
authors.forename: T E
authors.initials: TE
authors.lastname: Machen
mesh_terms: D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology; D006056:Golgi Apparatus / Q000502:physiology; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D015388:Organelles / Q000187:drug effects / Q000502:physiology*; D010902:Pituitary Gland / Q000502:physiology*; D010907:Pituitary Hormones / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology; D014407:Tumor Cells, Cultured; D025262:Vacuolar Proton-Translocating ATPases; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D002712:Chlorides; D010907:Pituitary Hormones; D011498:Protein Precursors; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases