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Substrate-level phosphorylation in isolated yeast mitochondria.

M Rigoulet, J Velours, B Guerin

    European Journal of Biochemistry
    |December 16, 1985
    PubMed
    Summary
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    Yeast mitochondria control substrate-level phosphorylation via 2-oxoglutarate supply, not respiration. This process maintains high internal phosphate potential, crucial for yeast energy metabolism.

    Area of Science:

    • Mitochondrial bioenergetics
    • Yeast metabolism
    • Enzyme kinetics

    Background:

    • Substrate-level phosphorylation (SLP) is a key ATP-generating pathway in mitochondria.
    • Understanding SLP control is vital for comprehending cellular energy homeostasis, especially in yeast.
    • Previous studies often focused on oxidative phosphorylation, leaving SLP regulation less explored.

    Purpose of the Study:

    • To investigate the activity and control mechanisms of substrate-level phosphorylation in isolated yeast mitochondria.
    • To identify the rate-limiting steps and regulatory components of SLP linked to 2-oxoglutarate oxidation.
    • To explore the bioenergetic implications of SLP in yeast.

    Main Methods:

    • Isolation of yeast mitochondria.
    • Measurement of ATP synthesis rates linked to 2-oxoglutarate oxidation.

    Related Experiment Videos

  • Determination of flux control coefficients for key mitochondrial components.
  • Analysis of endogenous nucleotide and phosphate levels and compartmentation.

    • Main Results:

    • Oligomycin-insensitive ATP synthesis via 2-oxoglutarate oxidation occurred at significant rates, comparable to oxidative phosphorylation.
    • Kinetic control was primarily localized to 2-oxoglutarate supply and the 2-oxoglutarate dehydrogenase complex.
    • Flux control coefficients for the respiratory chain, translocase, and phosphate carrier were near zero.
    • ADP was identified as the primary phosphate acceptor in endogenous nucleotide phosphorylation.
    • Evidence for metabolic compartmentation of nucleotides and phosphate within mitochondria was found.
    • A linear correlation existed between 2-oxoglutarate-linked ATP synthesis and internal phosphate potential (ΔG'p).

    Conclusions:

    • Substrate-level phosphorylation, particularly via 2-oxoglutarate, plays a significant role in yeast mitochondrial bioenergetics.
    • The regulation of SLP is mainly dependent on substrate availability and the activity of the 2-oxoglutarate dehydrogenase complex.
    • SLP alone can maintain a high internal phosphate potential, with important implications for yeast grown on fermentable substrates.