Minnie Wu, Juan Llopis, Stephen
Adams, J. Michael McCaffery, Terry Machen, Hsiao-Ping Moore and Roger
Tsien (2000). Use of organelle-targeted avidin and membrane-permeant,
fluorescent biotin to measure pH regulation in the ER and trans-Golgi.
Chemistry and Biology. 7, 197-209.
Mammalian organelles of the secretory pathway are of differing pH. The pH values
form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral,
the Golgi is mildly acidic and the secretory granules are more acidic still (pH 5). The mechanisms
that regulate pH in these organelles are still unknown.
Results: Using a novel
method, we tested whether differences in H+ ‘leak’ and/or counterion
conductances contributed to the pH difference between two secretory pathway
organelles. A pH-sensitive, membrane-permeable fluorescein–biotin was targeted
to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa
cells. In live, intact cells, ER pH (pHER) was 7.2 ± 0.2 and Golgi pH
(pHG) was 6.4 ± 0.3 and was dissipated by bafilomycin. Buffer
capacities of the cytosol, ER and Golgi were all similar (6–10 mM/pH). ER
membranes had an apparent H+ permeability three times greater than
that of Golgi membranes. Removal of either K+ or Cl− did
not affect ER and Golgi H+ leak rates, or steady-state pHG
Conclusions: The Golgi is
more acidic than the ER because it has an active H+ pump and fewer or
smaller H+ leaks. Neither buffer capacity nor counterion
permeabilities were key determinants of pHG, pHER or
ER/Golgi H+ leak rates.