Mechanisms of pH Regulation in the Regulated Secretory Pathway*

A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pHER = 7.4 ± 0.2, mean ± S.D.) to Golgi (pHG = 6.2 ± 0.4) to mature secretory granules (MSGs) (pHMSG= 5.5 ± 0.4). Golgi and MSGs required active H+v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H+ leak rates across each membrane. However, neither steady-state pHMSG nor rates of passive H+ leak were affected by Cl−-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pHMSG. Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl− conductances. Measurements of H+ leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical model to determine the H+ permeability (P H+) of each organelle membrane. We found that P H+ decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases inP H+ and successive increases in the active H+ pump density.