[Minnie Wu, Michael Grabe,
Stephen Adams, Roger Tsien, Hsiao-Ping.Moore and Terry Machen (2001).
Mechanisms of pH regulation in the regulated secretory pathway.
J. Biol. Chem. 276, 33027-33035.
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) (pH(ER) = 7.4 +/-
0.2, mean +/- S.D.) to Golgi (pH(G) = 6.2 +/- 0.4) to mature secretory granules
(MSGs) (pH(MSG) = 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 pH(MSG) 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 pH(MSG). 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 in P(H+) and
successive increases in the active H(+) pump density.