Edited by James Murray, PhD
Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how Abcam's Mitosciences range of research tools has contributed to this effort. Read the full list of 80 original research papers published in the last two months.
The oxidative phosphorylation system (OXPHOS), which resides within the inner mitochondria membrane structure termed cristae is comprised of the respiratory chain (Complexes I-IV) and ATP synthase (Complex V). Electron transfer by the respiratory chain is coupled to proton gradient generation across the inner membrane. The proton gradient is dissipated through the ATP synthase and generates ATP by a rotational catalytic mechanism. With so many component complexes transferring high energy intermediates it is then not surprising that the system is organized into quaternary respiratory supercomplexes and ATP synthase dimers which together form the respirasome. This assembly may confer increased stability, enhanced intermediate transfer efficiency and decreased electron and proton leakage , therefore limiting ROS generation. As a result impaired supercomplex formation may be critical in the etiology of multiple diseases and aging itself.
In a September article published in Cell, Cogliati et al. explored the relationship between the cristae and respiratory supercomplexes. They show that in a mouse model acute ablation of OPA1, a regulator of cristae shape, affects membrane organization, supercomplex assembly and complex I activity without affecting mtDNA levels. Only chronic OPA1 ablation resulted in decreased mtDNA levels which was therefore attributed as a consequence of cristae reorganization as a result of fusion inhibition. Conversely overexpression of OPA1 resulted in increased supercomplex formation, respiratory function and growth, again without an increase in mtDNA levels.
The authors propose that cristae remodeling impairs mitochondrial respiratory chain supercomplex assembly and mitochondrial function, which in turn precipitates apoptosis. Therefore disruption of OPA1 oligomerization is a significant player in mitochondria mediated intrinsic apoptotic pathway.
Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cell. 2013 Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado M, Cipolat S, Costa V, Casarin A, Gomes LC, Perales-Clemente E, Salviati L, Fernandez-Silva P, Enriquez JA, Scorrano L.
The mitochondria F1F0 ATP synthase is capable of dimerization and also oligomerization, a property which has been implicated in determining the structure and curvature of the cristae membranes. Two interfaces between oligomers have been proposed from work in yeast– 1) between homologues of subunits b and F6 in the peripheral stalk, both of which are essential for enzyme assembly, therefore hard to study by elimination, and 2) between subunit b and membrane subunits e and g. Homologues of subunits e and g are not essential in yeast and can therefore be more easily manipulated.
Recently, Habersetzer et al. examined dimerization and oligomerization in mammalian cells. To do this these researchers knocked down the levels of e and g in human cells with interfering RNA, showing that reduced levels of these proteins had more significant effects than those seen in yeast. They showed that reductions in e and g did not affect the accumulation of soluble F1 domain ATP synthase subunits but did affect the levels of stalk subunits OSCP, F1 and d. Using a control (scrambled) interfering RNA experiment, ATP synthase dimers and oligomers could be observed in blue-native and clear-native gel electrophoresis experiments, respectively. However, in a cell line where subunit e was knocked down, less stable and much diminished oligomers were observed, whilst dimers were completely absent.
The effects of subunit knock down were also observed in the structure of the mitochondrial network. Using a mitochondrial targeted GFP protein, a decrease in the proportion of cells with filamentous and hyper filamentous mitochondrial networks was observed and instead the mitochondria took either a fragmented or punctuate form. Using electron microscopy to examine mitochondrial ultra-structure the researchers found that cells where subunit e was knocked down had smaller spherical mitochondria that did not contain obvious cristae like membrane structures.
Depletion of e and g subunits increased the doubling time of cells and led to rapid acidification of cellular media indicating a shift in metabolism to a more glycolytic state. Respiratory rates and respiratory capacity were decreased consistent with this theory. Citrate synthase activities were similar between knock down and control experiments indicating similar mitochondrial enzymatic content however subunits of respiratory chain complexes III and IV, but not I and II, were significantly reduced in knock down cell lines.
In conclusion loss or reduction of these subunits in mammalian cells has a significant effect upon F1F0 ATP synthase assembly. These alterations result in decreased ATP synthase enzyme activity and have a direct effect upon respiratory chain activity. The authors propose that alterations to assembly, dimerization and oligomerization of the enzyme affect not only oxidative metabolism but also directly the cristae membrane structure itself.
Human F1F0 ATP Synthase, Mitochondrial Ultrastructure and OXPHOS Impairment: A (Super-) Complex Matter? PLoS One. 2013. Habersetzer J, Larrieu I, Priault M, Salin B, Rossignol R, Brèthes D, Paumard P.
OPA1 antibody [1E81D9] (ab119685)
MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411)
MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail (ab110413)
MitoProfile® Total OXPHOS Blue Native WB Antibody Cocktail (ab110412)
MitoTox™ Complex V OXPHOS Activity Microplate Assay (ab109907)
MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)