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 (pH ER ) was 7.2 ± 0.2 and Golgi pH (pH G ) 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 pH G and pH ER