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Subunit gamma-green fluorescent protein fusions are functionally incorporated into mitochondrial F1F0-ATP synthase, arguing against a rigid cap structure at the top of F1.

The Journal of Biological Chemistry

physiology, Adenosine Triphosphate, enzymology, cytology, Yeasts, metabolism, genetics, Recombinant Fusion Proteins, chemistry, Proton-Translocating ATPases, Protein Subunits, Protein Structure, Secondary, Protein Structure, Quaternary, Molecular Sequence Data, Models, Molecular, Mitochondria, Luminescent Proteins, Green Fluorescent Proteins, Fungal Proteins, Cell Fractionation, Cattle, Base Sequence, Animals, Amino Acid Sequence

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      Abstract

      We have investigated the question of the presence of a cap structure located at the top of the F(1) alpha(3)beta(3) hexamer of the yeast mitochondrial F(1)F(0)-ATP synthase complex. Specifically, we sought to determine whether the putative cap has a rigid structure and occludes the central shaft space formed by the alpha(3)beta(3) hexamer or alternatively whether the cap is more flexible permitting access to the central shaft space under certain conditions. Thus, we sought to establish whether subunit gamma, an essential component of the F(1) central stalk housed within the central shaft space and whose N and C termini would both lie beneath a putative cap, could be fused at its C terminus to green fluorescent protein (GFP) without loss of enzyme function. The GFP moiety serves to report on the integrity and location of fusion proteins containing different length polypeptide linkers between GFP and subunit gamma, as well as being a potential occluding structure in itself. Functional incorporation of subunit gamma-GFP fusions into ATP synthase of yeast cells lacking native subunit gamma was demonstrated by the ability of intact complexes to hydrolyze ATP and retain sensitivity to oligomycin. Our conclusion is that the putative cap structure cannot be an inflexible structure, but must be of a more flexible nature consistent with the accommodation of subunit gamma-GFP fusions within functional ATP synthase complexes.

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      Journal
      10.1074/jbc.M204556200
      12414811

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