Structure/function analysis of yeast mitochondrial ATP synthase subunit 8.
Subunit 8 of yeast mitochondrial ATP synthase is a small hydrophobic component of the membrane-associated F0 sector. Structure/function relations in subunit 8 were studied by focusing on three structural domains: a highly conserved NH2-terminal region, a central hydrophobic region (previously suggested to be a transmembrane stem), and a COOH-terminal region bearing a conserved array of three positively charged residues. A combined approach was used, which encompasses site-directed mutagenesis, in vitro import and assembly tests, and an in vivo allotopic expression system (using host cells unable to synthesise subunit 8 in mitochondria). The results indicate that the NH2-terminal region of subunit 8 is involved functionally in the F0 sector. As the central hydrophobic region can functionally tolerate the introduction of multiple, positively charged residues (which abolishes the proteolipid solubility characteristics of the entire subunit), the role of this hydrophobic region as a transmembrane stem is brought into question. Each of the three positively charged residues toward the COOH-terminus of subunit 8 is required for the efficient assembly of this subunit into the F0 sector. Removal of the more proximal charged residues Arg37 or Arg42 has a more severe impact on subunit 8 assembly than does removal of the most distal residue Lys47 in terms of both in vitro import and assembly as well as the ability of the subunit 8 variant to function in mitochondrial ATP synthase in vivo.
Devenish
R J
RJ
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Papakonstantinou
T
T
Galanis
M
M
Law
R H
RH
Linnane
A W
AW
Nagley
P
P
D000595:Amino Acid Sequence; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases