Organelle pH studies using targeted avidin and fluorescein–biotin

Abstract

Background: 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 and pHER.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.