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ATP Driven Pumps III: V-type Pumps01:30

ATP Driven Pumps III: V-type Pumps

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V-type pumps are ATP-driven pumps found in the vacuolar membranes of plants, yeast, endosomal and lysosomal membranes of animal cells, plasma membranes of a few specialized eukaryotic cells, and some prokaryotes. They are also known as the V1Vo-ATPase, that couple ATP hydrolysis to transport protons against a concentration gradient.
The peripheral or cytosolic V1 domain with eight subunits is involved in ATP hydrolysis. The integral or transmembrane V0 domain containing at least five subunits...
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Related Experiment Video

Updated: Dec 24, 2025

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
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Role of V-ATPase a3-Subunit in Mouse CTL Function.

Praneeth Chitirala1, Keerthana Ravichandran1, Claudia Schirra1

  • 1Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany.

Journal of Immunology (Baltimore, Md. : 1950)
|April 10, 2020
PubMed
Summary

The vacuolar adenosine triphosphatase a3-subunit acidifies cytotoxic granules (CG) in T cells. This acidification is crucial for CG maturation, transport to the immune synapse, and effective target cell killing.

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Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Cytotoxic T lymphocytes (CTLs) kill target cells by releasing cytotoxic proteins from cytotoxic granules (CG).
  • CGs have an acidic internal pH essential for processing cytotoxic proteins like granzymes and perforin.
  • The precise role of this acidic pH in CG maturation, transport, and fusion remains unclear.

Purpose of the Study:

  • To investigate the role of the vacuolar-type (H+) -adenosine triphosphatase a3-subunit in cytotoxic granule (CG) acidification.
  • To determine the impact of a3-subunit function on CG maturation, transport, and fusion with the target cell.

Main Methods:

  • Utilized primary murine CTLs and a novel CG-specific pH indicator, ClopHensorN(Q69M), to measure luminal pH.
  • Employed knockdown of the a3-subunit to assess its functional importance.
  • Applied total internal reflection fluorescence microscopy and superresolution microscopy to analyze CG fusion and interaction with microtubules.
  • Used electron and structured illumination microscopy to examine CG morphology.

Main Results:

  • The a3-subunit of vacuolar-type (H+) -adenosine triphosphatase is essential for maintaining an acidic pH (6.1) within CGs.
  • Knockdown of the a3-subunit significantly reduced target cell killing and CG fusion (>50%).
  • Reduced CGs at the immune synapse and impaired interaction with the microtubule network were observed upon a3-subunit knockdown.
  • CG morphology, including diameter and density, was altered, though the total number of CGs per CTL remained unchanged.

Conclusions:

  • The a3-subunit is critical for cytotoxic granule acidification.
  • Acidification by the a3-subunit is vital for proper CG maturation and efficient transport to the immune synapse.
  • This process is essential for effective CTL-mediated cytotoxicity.