Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme |
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Authors: | Petr Pecina Hana N?sková Vendula Karbanová Vilma Kaplanová Tomá? Mrá?ek Josef Hou?těk |
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Institution: | Department of Bioenergetics, Institute of Physiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic |
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Abstract: | The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150–400?kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase. |
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Keywords: | 1 catalytic part of ATP synthase o membrane embedded part of ATP synthase 1 1 1 1 F6 ATP synthase subunit coupling factor 6 OSCP ATP synthase subunit oligomycin sensitivity conferring protein o o M membrane potential of mitochondrial membrane ATP synthase Deficiency Knockdown γ and δ subunits Subunit c aggregates |
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