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

• Published in: The Journal of cell biology Vol. 221; no. 3; p. 1
• Main Authors: Maxson, Michelle E, Abbas, Yazan M, Wu, Jing Ze, Plumb, Jonathan D, Grinstein, Sergio, Rubinstein, John L
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 07.03.2022
• Subjects: Acidification; Adenosine triphosphatase; Animals; Bacterial Proteins - metabolism; Chimeras; Density; Fluorescence; H+-transporting ATPase; HeLa Cells; Humans; Hydrogen; Hydrogen-Ion Concentration; Legionella - metabolism; Legionnaires' disease bacterium; Localization; Lysosomes; Lysosomes - metabolism; Membrane proteins; Membrane trafficking; Membranes; Mice; Organelles; Phagosomes - metabolism; Physiology; Plasmids; Protein transport; Proteins; Rats; RAW 264.7 Cells; Reagents; Saccharomyces cerevisiae - metabolism; Secretory vesicles; Solute transport; Vacuolar Proton-Translocating ATPases - metabolism
• DOI: 10.1083/jcb.202107174
• ISSN: 0021-9525

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 SidK1-278, and labeled recombinant SidK1-278 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.