Determinants of the pH of the Golgi complex.
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.
Schapiro
F B
FB
Cell Biology Programme, Research Institute, The Hospital for Sick Children and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
Grinstein
S
S
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
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