We determined the H+ and Ca2+ uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca2+ sequestering is due to Ca2+/H+ antiporter(s) localized to secretory pathway organelles while Ca2+-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca2+ and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca2+ signaling in a differential and local manner.