Solution structure of subunit a, a₁₀₄₋₃₆₃, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation

J Bioenerg Biomembr. 2012 Jun;44(3):341-50. doi: 10.1007/s10863-012-9442-3. Epub 2012 May 5.

Abstract

The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa₁₀₄₋₃₆₃ of the N-terminal domain (SCa₁₋₃₆₃) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa₁₀₄₋₃₆₃ shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A₁A₀ ATP synthase from Pyrococcus furiosus, revealing that the SCa₁₀₄₋₃₆₃ fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa₁₋₃₆₃, and to determine the localization of the N- and C-termini in SCa₁₀₄₋₃₆₃, the C-terminal truncated form SCa₁₀₆₋₃₂₄ was produced and analyzed by SAXS. Comparison of the SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ shapes showed that the additional loop region in SCa₁₀₄₋₃₆₃ consists of the C-terminal residues. Whereas SCa₁₀₄₋₃₆₃ is monomeric in solution, SCa₁₀₆₋₃₂₄ forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Circular Dichroism
  • GTPase-Activating Proteins / chemistry
  • GTPase-Activating Proteins / metabolism
  • Guanine Nucleotide Exchange Factors / chemistry
  • Guanine Nucleotide Exchange Factors / metabolism
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Subunits
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Solutions / chemistry
  • Vacuolar Proton-Translocating ATPases / chemistry*
  • Vacuolar Proton-Translocating ATPases / genetics
  • Vacuolar Proton-Translocating ATPases / metabolism

Substances

  • GTPase-Activating Proteins
  • Guanine Nucleotide Exchange Factors
  • Peptide Fragments
  • Protein Subunits
  • Sec7 guanine nucleotide exchange factors
  • Solutions
  • cytohesin-2
  • Vacuolar Proton-Translocating ATPases