No abstract

We have used the tetrameric nature of the fluorescent protein DsRed to cross-link F1FO-ATPase complexes incorporating a subunit γ-DsRed fusion protein in vivo. Cells expressing such a fusion protein have impaired growth relative to control cells. Strikingly, fluorescence microscopy of these cells revealed aberrant mitochondrial morphology. Electron microscopy of cell sections revealed the absence of cristae and multiple layers of unfolded inner mitochondrial membrane. Complexes recovered from detergent lysates of mitochondria were present largely as tetramers. Co-expression of `free' DsRed targeted to the mitochondria reduced F1FO-ATPase oligomerisation and partially reversed the impaired growth and abnormal mitochondrial morphology. We conclude that the correct arrangement of F1FO-ATPase complexes within the mitochondrial inner membrane is crucial for the genesis and/or maintenance of mitochondrial cristae and morphology. Our findings further suggest that F1FO-ATPase can exist in oligomeric associations within the membrane during respiratory growth.

Evidence suggests membrane bound F(1)F(0)-ATPase complexes form stable associations such that dimers can be retrieved from detergent lysates of mitochondria isolated from a range of sources including algae, higher plants, yeast and bovine heart, and plant chloroplasts. The physiological relevance of these interactions is not clear but may be connected with the formation and structure of mitochondrial cristae. We sought to demonstrate, in vivo, the association of F(1)F(0)-ATPases in yeast cells co-expressing two b subunits each fused at its C-terminus to a GFP variant appropriate for fluorescence resonance energy transfer (FRET; BFP as the donor and GFP as the acceptor fluorophore). Both subunit b-GFP and b-BFP fusions were assembled into functional complexes. FRET was observed from enzyme complexes in molecular proximity in respiring cells providing the first demonstration of the association, in vivo, of F(1)F(0)-ATPase complexes. Moreover, FRET was observed within cells lacking the dimer specific subunit e, indicating structured associations can occur within the inner membrane in the absence of subunit e.

No abstract

The TPM/oxycodone patch delivered oxycodone to the site of perceived pain in subjects suffering from PHN, but did not provide analgesia for the broad PHN indication.