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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.
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Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
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Interaction of yeast V-ATPase with TLDc protein Rtc5p.

Md Murad Khan1, Roshanak Ebrahimi1, Rebecca A Oot1

  • 1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.

Biorxiv : the Preprint Server for Biology
|June 6, 2025
PubMed
Summary

Rtc5p aids in assembling the vacuolar H+-ATPase (V-ATPase) enzyme from its parts. Structural studies reveal its mechanism, suggesting multiple V-ATPase assembly pathways exist.

Keywords:
Protein structureReversible disassemblyRtc5pTLDc domainVacuolar H+-ATPase

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The vacuolar H+-ATPase (V-ATPase) regulates cellular processes via reversible disassembly into V1 and Vo subcomplexes.
  • Understanding the molecular mechanisms of V-ATPase assembly is crucial but remains poorly understood.
  • Oxr1p is known to be essential for V-ATPase disassembly in vivo.

Purpose of the Study:

  • To elucidate the role of Rtc5p, a TLDc domain protein, in the reversible assembly of V-ATPase.
  • To investigate the molecular mechanisms by which Rtc5p facilitates V-ATPase assembly.

Main Methods:

  • In vitro assembly assays using purified V1 and Vo subcomplexes.
  • Cryo-electron microscopy (CryoEM) to determine structural details of Rtc5p-V-ATPase interactions.
  • Phenotypic analysis of Rtc5p function in yeast.

Main Results:

  • Rtc5p promotes the assembly of functional holo V-ATPase from purified V1 and Vo subcomplexes in vitro.
  • CryoEM structures reveal Rtc5p binds the V1-B subunit and inserts an alpha-helix into the catalytic hexamer, potentially opening a second catalytic site.
  • Rtc5p is not essential for glucose-driven V-ATPase assembly in vivo, unlike Oxr1p.

Conclusions:

  • Rtc5p functions as an assembly factor for the V-ATPase, distinct from the disassembly role of Oxr1p.
  • The structural data provides insights into the mechanism of V-ATPase assembly mediated by Rtc5p.
  • The findings suggest the existence of multiple, potentially parallel, pathways for V-ATPase assembly and regulation in vivo.