The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

For Fig.1b, Anti-V5 and Anti-HA blots are shown. Size fractions of Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG from HS extract in a buffer containing 0 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.1c, Anti-FLAG and Anti-HA blots are shown below. Size fractions of Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG from LS extract in a buffer containing 0 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.

Anti-V5
Anti-V5 Input Input For Fig.1d, Anti-FLAG and Anti-HA blots are shown below. Size fractions of Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG from HS extract in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.1d, Anti-V5 blots are shown below. Size fractions of Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG from HS extract in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.1e, Anti-FLAG blots are shown below. Size fractions of Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG from LS extract in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.1e, Anti-HA blots are shown below. Size fractions of Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG from LS extract in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.2a, Anti-FLAG blots are shown below. Size fractions of FLAG purified Pdb1-5xFLAG in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.2b, Anti-FLAG blots are shown below. Size fractions of FLAG purified Lpd1-5xFLAG in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.2d, Anti-FLAG blots are shown below. Size fractions of FLAG purified Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG in a buffer containing 0 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.2d, Anti-V5 blots are shown below. Size fractions of FLAG purified Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG in a buffer containing 0 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.2d, Anti-HA blots are shown below. Size fractions of FLAG purified Pdb1-3xHA/Lat1-V5/Lpd1-5xFLAG in a buffer containing 0 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.3a, Anti-mE1pa blots are shown below. Size fractions of HEK293T lysates in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
α α For Fig.3a, Anti-mE2p and Anti-mE1pb blots are shown below. Size fractions of HEK293T lysates in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated. β β For Fig.3a, Anti-mE3 blots are shown below. Size fractions of HEK293T lysates in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated.
For Fig.3b, Anti-HA blots are shown below. Size fractions of Halo-purified PDHA1-HA ectopically expressing in HEK293T lysates in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every 500 μl fraction indicated. For S4a, Anti-mE1pα blots are shown below. Size fractions of HEK293T lysates in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every other 500 μl fraction indicated.
α For S4a, Anti-Baf155 blots are shown below. Size fractions of HEK293T lysates in a buffer containing 350 mM NaCl with the elution volumes in the SEC via Superose 6 column for every other For S6, Anti-mE2p blots are shown. Size fraction of affinity purified mE1pα (PDHA1-HA) from HEK293T cells in a buffer containing 0 mM NaCl with the Elution volumes in the SEC via Superose 6 column for every 500 µl fraction indicated.

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For S6, Anti-HA blots are shown. Size fraction of affinity purified mE1pα (PDHA1-HA) from HEK293T cells in a buffer containing 0 mM NaCl with the Elution volumes in the SEC via Superose 6 column for every 500 µl fraction indicated.
Anti-HA Anti-HA .. --For S7, Anti-FLAG blots are shown. Same amount of samples containing FLAG purified pkp1/Pdb1-5xFLAG from the catalytic activity assay for 50 mM and 350 mM NaCl conditions was loaded