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Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
13:55

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

Published on: April 19, 2013

The 31-kDa polypeptide is an essential subunit of the vacuolar ATPase in Saccharomyces cerevisiae

F Foury1

  • 1Unité de Biochimie Physiologique, Université de Louvain, Louvain-la-Neuve, Belgium.

The Journal of Biological Chemistry
|October 25, 1990
PubMed

Insights

The VMA4 gene is essential for vacuolar ATPase function in yeast, impacting growth and pigment accumulation. This finding highlights conserved vacuolar ATPase components from yeast to mammals.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The vacuolar ATPase (V-ATPase) is a crucial proton pump found in eukaryotic cells, responsible for maintaining acidic organelles.
  • The VMA4 gene's role and its specific contribution to V-ATPase function were not fully elucidated.
  • Understanding V-ATPase subunit function is vital for comprehending cellular homeostasis and disease mechanisms.

Purpose of the Study:

  • To characterize the function of the VMA4 gene and its encoded polypeptide in yeast.
  • To determine if VMA4 is an essential component of the vacuolar ATPase.
  • To investigate the regulatory elements and gene linkage associated with VMA4.

Main Methods:

  • Gene inactivation of the chromosomal VMA4 gene using deletion and URA3 insertion.
  • Phenotypic analysis of null vma4 haploid mutants, including growth rate, temperature sensitivity, and pigment accumulation.
  • Biochemical assays to detect bafilomycin-sensitive ATPase activity in vacuolar fractions.
  • Analysis of VMA4 and MIP1 gene transcription and promoter regions.

Main Results:

  • Null vma4 mutants exhibited significantly slowed growth, particularly in non-acidic conditions, and displayed cold and thermo-sensitive phenotypes.
  • Mutants showed poor growth on glycerol and failed to accumulate the red pigment in ade2 strains, indicating vacuolar dysfunction.
  • No bafilomycin-sensitive ATPase activity was detected in vacuolar fractions of null mutants.
  • VMA4 is divergently transcribed from the MIP1 gene, with conserved regulatory elements suggesting TUF protein involvement.

Conclusions:

  • The VMA4 polypeptide is an essential component of the yeast vacuolar ATPase, conserved across species.
  • VMA4 plays a critical role in cellular growth, division, and vacuolar acidification.
  • The VMA4 gene's transcription is likely regulated by the TUF protein, linking it to cellular growth and division pathways.

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