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Expression and function of the mouse V-ATPase d subunit isoforms.

Tsuyoshi Nishi1, Shoko Kawasaki-Nishi, Michael Forgac

  • 1Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA.

The Journal of Biological Chemistry
|September 10, 2003
PubMed
Summary

Researchers discovered a new mouse V-ATPase d2 isoform. This isoform functions differently from d1, particularly in yeast complementation and V-ATPase assembly, suggesting a role in proton transport and ATP hydrolysis coupling.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The V-ATPase is a crucial proton pump involved in various cellular functions.
  • The d subunit is a component of the V-ATPase, with known isoforms in yeast (Vma6p) and mouse (d1).

Purpose of the Study:

  • To identify and characterize a novel isoform of the mouse V-ATPase d subunit.
  • To investigate the functional differences between the d1 and d2 isoforms and their roles in V-ATPase assembly and activity.

Main Methods:

  • cDNA identification and sequencing.
  • Reverse transcriptase-PCR (RT-PCR) for expression analysis.
  • Yeast complementation assays at different temperatures.
  • Measurement of V-ATPase assembly, ATPase activity, and proton transport.

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Main Results:

  • A novel mouse V-ATPase d subunit isoform, d2, was identified, sharing 67% identity with d1 and 42% with yeast Vma6p.
  • d2 is primarily expressed in the kidney.
  • While both d1 and d2 can complement yeast VMA6 disruption, d2 functions optimally at 25°C, unlike d1 which requires 30°C.
  • d2 incorporation leads to partial V-ATPase assembly, reduced ATPase activity, and lower proton transport compared to Vma6p.
  • Kinetic analysis shows d1-containing complexes have a higher coupling ratio of proton transport to ATP hydrolysis than Vma6p-containing complexes.

Conclusions:

  • The mouse V-ATPase d2 isoform exhibits distinct functional properties compared to d1 and yeast Vma6p.
  • The d subunit, including the novel d2 isoform, appears to play a role in regulating the coupling efficiency between proton transport and ATP hydrolysis in the V-ATPase complex.