Abstract
The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.
Highlights
The F1F0-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes
We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein
Molecular sizing analysis, either gel filtration or native gel electrophoresis of the ATP synthase complexes solubilized from mitochondrial membranes with a mild detergent, demonstrated that the ATP synthase can be isolated as a complex whose mass (ϳ1000 kDa) was consistent with that of a dimeric complex [5, 6, 9, 14]
Summary
Yeast Strains and Growth Conditions—Yeast strains used in this study were wild-type (WT) W303–1A (Mat a, leu, trp, ura, his, ade2) and the su g null mutant, ⌬su g (W303-1A, leu, trp, ura, ade, ATP20::HIS3) [6]. Mitochondria were isolated from the resulting yeast strains, which had been grown in YP (yeast extract, peptone) supplemented with 0.5% lactate and galactose (2%, YP-galactose medium) or glycerol (3%, YPG medium), as indicated [26]. Mitochondria were isolated from each of the transformants, and the expression and mitochondrial localization of the Su g derivatives were verified by Western blotting using an antibody specific for the HA epitope (Covance Research Products) or Su g antiserum [6]. Growth Curve Analysis—All strains were initially grown overnight at 30 °C in YPG medium supplemented with 0.2% galactose and histidine (0.06 mg/ml) and leucine (0.26 mg/ml). Rho0/rhoϪ Cell Conversion Detection Assay—All yeast strains were maintained on YPG plates and were used to inoculate YP-lactate (0.5% lactate) supplemented with 2% galactose and allowed to grow overnight at 30 °C. Clear native-PAGE analysis of F1F0-ATP synthase complexes after solubilization with digitonin was performed essentially as described previously [15]
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