Vacuolar Atpase

Vacuolar ATPase (V-ATPase) is a multisubunit proton pump that uses ATP to acidify intracellular compartments and, in some cells, the extracellular space. Its cytosolic V1 domain hydrolyzes ATP, driving rotation that powers the membrane-embedded V0 domain to translocate protons across lipid membranes. This proton gradient supports endosomal and lysosomal function, protein trafficking, membrane remodeling, and activation of pH-dependent enzymes. In developmental biology, V-ATPase activity helps regulate cell signaling, tissue organization, organelle maturation, and morphogenetic processes, while defects in its subunits or assembly can disrupt development and cause disease.

Vacuolar Atpase - Related Videos

Research

JoVE Journal - Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

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Cited by 27 •

2016

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

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Cited by 19 •

2015

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages

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2025

This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin,...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

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Cited by 40 •

2013

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Research

JoVE Journal - Immunology and Infection
Free Sample

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

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Cited by 22 •

2013

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

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