Detection and quantification of the vacuolar H ATPase using the Legionella effector protein SidK

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar ATPases (V ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive nor specific to test this hypothesis. We introduce a new probe to localize and quantify V ATPases in eukaryotic cells. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1 278, and labeled recombinant SidK1 278 with AlexaFluor-568 to visualize and quantify V ATPases with high specificity in live and fixed cells, respectively. We show that V ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on the subcellular localization of the lysosome.