title: Determinants of the pH of the Golgi complex.
abstract: The factors contributing to the establishment of the steady state Golgi pH (pH(G)) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K(+) in the lumen of the Golgi using a null point titration method. The [K(+)] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH(G). Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H(+) pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH(G). The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH(G). In fact, active H(+) pumping was detectable even below the resting pH(G). A steady state pH was attained when the rate of pumping was matched by the passive backflux of H(+) (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H(+)/cation antiporters or symporters were found to contribute to the net loss of H(+) from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn(2+), resembling the H(+) conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H(+) pump determines the pH at which the Golgi lumen attains a steady state.
authors.affiliation: Cell Biology Programme, Research Institute, The Hospital for Sick Children and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
authors.forename: F B
authors.initials: FB
authors.lastname: Schapiro
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011188:Potassium / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013816:Thermodynamics; D025262:Vacuolar Proton-Translocating ATPases; D014709:Vero Cells; D017967:Zinc Compounds / Q000494:pharmacology
chemical_list: D002712:Chlorides; D005456:Fluorescent Dyes; D017967:Zinc Compounds; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C016837:zinc chloride; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium