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J. Biol. Chem., Vol. 275, Issue 28, 21025-21032, July 14, 2000
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From the Cell Biology Programme, Research Institute, The Hospital
for Sick Children and the Department of Biochemistry, University of
Toronto, Toronto, Ontario M5G 1X8, Canada
The factors contributing to the establishment of
the steady state Golgi pH (pHG) 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 pHG. 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 pHG. The V-ATPase does not reach
thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pHG. In fact, active
H+ pumping was detectable even below the resting
pHG. 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
Zn2+, 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.
Determinants of the pH of the Golgi Complex*
*
This work was supported by the Canadian Cystic Fibrosis
Foundation and the Medical Research Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
International Scholar of the Howard Hughes Medical Institute.
Recipient of a Medical Research Council Distinguished Scientist Award.
Current holder of the Pitblado Chair in Cell Biology. To whom
correspondence should be addressed: Cell Biology Programme, Hospital
for Sick Children, 555 University Ave., Toronto, ON M5G 1X8, Canada.
Tel.: 416-813-5727; Fax: 416-813-5028; E-mail:
sga@sickkids.on.ca.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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