Membrane-coating granules are acidic organelles which possess proton pumps

Lysosomes are by definition organelles that maintain an internal acidic pH and contain hydrolytic enzymes. Membrane-coating granules contain a battery of hydrolytic enzymes, in addition to their lamellar discs, and are therefore commonly assumed to be lamellate lysosomes. Although there are data confirming the existence of enzymes in membrane-coating granules, there is no direct evidence to suggest that their internal pH is acidic. As part of a wider program on their role in desquamation, our aim was to determine whether membrane-coating granules are indeed acidic and possess proton pumps. Chloroquine and monensin were selected as the pH markers because both induce swelling of acidic organelles. In four repeat experiments dermatome slices of pig ear skin (2 mm2 x 0.5 mm) were incubated as organ cultures either alone (control) or with 1 mM chloroquine or 25 microM monensin. Ultrastructural observations revealed no swelling in control specimens. In contrast, the inclusion of chloroquine or monensin caused swelling of specific organelles including membrane-coating granules, lysosomes, and trans elements of Golgi stacks, but not mitochondria, rough endoplasmic reticulum, or nuclear envelopes. Swelling of membrane-coating granules and the other organelles was prevented by pretreatment with N,N'-dicyclohexylcarbodiimide, a known inhibitor of lysosomal H+ ATPase activity. These findings suggest that membrane-coating granules actively maintain an acidic interior with the aid of proton pumps. Furthermore, membrane-coating granules are heterogeneous because swelling of the whole population did not commence simultaneously. However, it remains to be determined whether this heterogeneity reflects variations in membrane-coating granule pH, leakiness of their membranes to cations, or the number or activity of their proton pumps.