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 H+ 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 or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. 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 SidK(1-278), and labeled recombinant SidK(1-278) with Alexa Fluor 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 their subcellular localization.

Automated summary

This study shows that proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for the acidic environment in secretory and endocytic organelles. The researchers developed a new probe derived from SidK protein to visualize and quantify V-ATPases with high specificity. They found that the density of V-ATPases increases during phagosome maturation and their distribution varies in lysosomes depending on their subcellular localization.