Manipulations in the Peripheral Stalk of the Saccharomyces cerevisiae F1F0-ATP Synthase

• Published in: The Journal of biological chemistry Vol. 286; no. 12; pp. 10155 - 10162
• Main Authors: Welch, Amanda K., Bostwick, Caleb J., Cain, Brian D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-03-2011; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; ATP Synthase; Bioenergetics; Escherichia coli - enzymology; Escherichia coli - genetics; F1Fo ATPase; Membrane Proteins; Mitochondria - chemistry; Mitochondria - enzymology; Mitochondria - genetics; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sequence Deletion; Site-directed Mutagenesis; Yeast; Membrane Proteins; Site-directed Mutagenesis; ATP Synthase; F1Fo ATPase; Yeast
• DOI: 10.1074/jbc.M110.213447
• EISSN: 1083-351X
• ISSN: 0021-9258

The Saccharomyces cerevisiae F1F0-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F1. In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F1F0-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F1F0-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.