Fig. 1.

The principle of leak detection. A dye, shown in pink, is constantly pumped across the plasma membrane. Top left: the volume enclosed by a membrane invagination is connected to the extracellular environment. Top right: enzymes delivered before a seal develops will leak away. Bottom left: the membrane invagination is sealed off from the environment, causing the dye to accumulate. Bottom right: enzymes delivered once the dye accumulates will not leak away. This principle applies both to endocytic as well as secretory organelles that are transiently connected to the extracellular environment. Eukaryotes universally use protons as the leak-detection dye, pumped by V-ATPases