Each of three positively-charged amino acids in the C-terminal region of yeast mitochondrial ATP synthase subunit 8 is required for assembly

The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

“…The lysine at position 37 and two arginines at positions 42 and 47 were not substituted as each of these residues contributes to the Cterminal positively charged region required for assembly of Y8 into mtATPase (28). To test the functionality in vivo of Y8 cysteine variants, M31(aap1 Ϫ ) cells expressing each single cysteine variant were plated onto medium containing ethanol as the sole carbon source.…”

“…M31 cells were transformed by the method of Klebe et al (38) with the pPD72 expression vector that carries a functional ADE1 gene (28). Transformants displaying the Ade ϩ phenotype were selected and restoration of respiratory-competent phenotype by the expressed Y8 cysteine variants was assessed by testing growth on medium containing ethanol as the sole carbon source.…”

“…In a previous study, analysis of allotopically expressed Y8 variants having a cysteine substitution at the N-or C-terminal residue of Y8 demonstrated that the CHD spans the inner mitochondrial membrane with N out -C in orientation (37). Here we have combined cysteine-scanning mutagenesis with allotopic expression to construct a series of single cysteine replacements encompassing the entire length of the Y8 protein (excluding three residues within the C-terminal positively charged region whose replacement would abrogate function (28)). Without exception each cysteine replacement was tolerated and all of the expressed Y8 variants restored mtATPase function in cells lacking endogenous Y8, demonstrating that none of the substituted amino acid residues of Y8 are essential for either ATP synthesis/hydrolysis activity or proton flow.…”

“…Allotopic expression (24), whereby a mitochondrial gene is recoded for nuclear expression with subsequent delivery of the protein back to mitochondria, has enabled our laboratory to undertake a detailed molecular genetic approach to investigate the structure and function of Y8. Detailed studies on genetically modified Y8 variants, allotopically expressed in yeast lacking endogenous Y8, have suggested that the N-terminal motif of Y8 plays a functional role in mtATPase (25)(26)(27) while the positively charged amino acid region at the C terminus of Y8 is involved in both the assembly and function of the F 0 sector (27)(28)(29). A surprising feature of Y8 is the functional accommodation of charged amino acid residues within the CHD (30 -33).…”

Single Copies of Subunits d, Oligomycin-sensitivity Conferring Protein, and b Are Present in the Saccharomyces cerevisiaeMitochondrial ATP Synthase

“…One may further speculate that the function of the small membrane integral and hydrophobic subunit 8 replaces the role of the N-terminal region of subunit b. The C-terminal portion of subunit 8 includes three positively charged residues and extends into the matrix and would be available to make contact with subunit d (24). Thus, in yeast, the combination of the hydrophobic integral membrane protein, subunit 8, and subunit d may serve the function of the second copy of subunit b.…”