Efimova 2017 MiP2017
Mitochondrial F1Fo ATP synthase is the major generator of ATP in eukaryotic cell. It is a multi-subunit enzyme with two-genome origins, which requires carefully orchestrated biogenesis. While the general assembly scheme had been proposed, there is still a lack of detailed information defining the intermediates during the middle and final stages of the enzyme assembly. In our laboratory, we have recently established a number of transgenic models deficient for the subunits of ATP synthase: a, A6L, ε, γ, δ, DAPIT, MLQ and also for a TMEM70 assembly factor, where presence of noncanonical intermediates can be observed. The aim of this project is to characterize and describe the individual assembly intermediates using the plethora of our cellular and animal models. Our results should improve our understanding of complex V maintenance and assembly and its supramolecular organization.
Visualization of F1Fo ATP synthase assembly intermediates was performed by electrophoretic analysis (SDS-, BN-, CN- and hrCN1-PAGE/WB) combined with western blot detection or ATPase in-gel activity assay. We also performed complex V immunocapture by IP and pulse-chase metabolic labeling of mitochondrial proteosynthesis. We are working on adaptation of the technique of affinity purification through IF1 inhibitor protein [1,2] in order to isolate individual ATP-synthase subassemblies and study their subunit composition using mass spectrometry and electrophoretic techniques.
Preliminary results revealed a presence of non-canonical intermediates of ATP synthase biogenesis under various electrophoretic conditions. Subunit composition of the observed intermediates in TMEM70 and DAPIT knockout models remains unknown. Moreover, purification of those entities by immunocapture techniques in their native forms was unsuccessful. Pulse-chase metabolic labeling allowed us to identify differences in the stability of mtDNA encoded subunits a and A6L between control HEK293 and MLQ -/- cells. However, apart from the separation of solubilizates from whole mitochondria on native gels, we lack the information about the presence of potential subassemblies. As the first step of IF1 based affinity purification, the sequence of murine IF1 protein fused with C-terminal gluthatione-S-transferase (GST) was cloned into the expression plasmid pET containing C-terminal hexahistidine tag. The recombinant protein was then expressed in E. coli BL21 (DE3 strain). We are currently going through the optimization of conditions for recombinant IF1 protein solubilization and purification. In the next step we will optimize conditions for ATP synthase solubilization from mitochondrial preparations for the following affinity purification.
In conclusion, we detected several non-canonical intermediates of ATP synthase biogenesis on various models of complex V deficiencies. We now aim to analyze subunit composition of the enzyme and its intermediates by their purification via IF1 affinity chromatography.
Labels: MiParea: Genetic knockout;overexpression
Organism: Human, Eubacteria Tissue;cell: HEK
Affiliations and support
- Dept Bioenergetics, Inst Physiology, Czech Academy Sciences, Prague, Czech Republic. - [email protected]
- Supported by the Czech Ministry of Health (16-33018A).
- Bason JV, Runswick MJ, Fearnley IM, Walker JE (2011) Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase. J Mol Biol 406:443–53.
- Runswick MJ, Bason JV, Montgomery MG, Robinson GC, Fearnley IM, Walker JE (2013) The affinity purification and characterization of ATP synthase complexes from mitochondria. Open Biol 3:120-160.