Abstract
Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution / genetics
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Cross-Linking Reagents / chemistry
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Cysteine / genetics
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Enzyme Activation
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Intracellular Membranes / enzymology
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Lysine / genetics
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Maleimides / chemistry
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Mitochondria / enzymology
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Mitochondrial Proton-Translocating ATPases / chemistry*
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Mitochondrial Proton-Translocating ATPases / genetics
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Mitochondrial Proton-Translocating ATPases / physiology*
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Molecular Sequence Data
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / physiology
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Protein Processing, Post-Translational
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Protein Subunits / chemistry*
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Protein Subunits / genetics
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Protein Subunits / physiology*
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / physiology*
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Sodium-Potassium-Exchanging ATPase / chemistry
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Succinimides / chemistry
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Vacuolar Proton-Translocating ATPases / chemistry
Substances
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Cross-Linking Reagents
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Maleimides
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Peptide Fragments
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Protein Subunits
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Saccharomyces cerevisiae Proteins
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Succinimides
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VMA8 protein, S cerevisiae
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1,6-bismaleimidohexane
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ATP14 protein, S cerevisiae
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F1F0-ATP synthase
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Vacuolar Proton-Translocating ATPases
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Mitochondrial Proton-Translocating ATPases
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Sodium-Potassium-Exchanging ATPase
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dithiobis(succinimidylpropionate)
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Lysine
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Cysteine
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N,N'-2-phenylenedimaleimide