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 (PH) of each organelle membrane. We found that PH decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases in PH and successive increases in the active H pump density.