Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme

https://doi.org/10.1016/j.bbabio.2018.02.007Get rights and content
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Highlights

  • Knockdown of γ or δ subunit decreases the content of mammalian ATP synthase.

  • No incomplete F1 or F1-c oligomer assembliess are detected.

  • Unused Fo subunits c accumulate as aggregates, not containing other enzyme subunits.

  • Increased content of IF1

  • Respiration analysis reveals decreased ADP-phosphorylating capacity but not increased proton leak.

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.

Abbreviations

F1
catalytic part of ATP synthase
Fo
membrane embedded part of ATP synthase
IF1
F1 inhibitor protein
F1 α
F1 β, F1 γ, F1 δ, F1 ε - F1 subunits α, β, γ, δ, and ε
F6
ATP synthase subunit coupling factor 6
OSCP
ATP synthase subunit oligomycin sensitivity conferring protein
Fo a
Fo d, Fo c, Fo subunits a, d and c
ΔΨM
membrane potential of mitochondrial membrane

Keywords

ATP synthase
Deficiency
Knockdown
γ and δ subunits
Subunit c aggregates

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