Yeast Pyruvate Dehydrogenase Complex Research Articles

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation.

The mitophagic degradation of mitochondrial matrix proteins in Saccharomyces cerevisiae was previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates.

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.

Subunit and Catalytic Component Stoichiometries of an in Vitro Reconstituted Human Pyruvate Dehydrogenase Complex

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772-19780).

Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases

The activity of yeast pyruvate dehydrogenase complex is regulated by reversible phosphorylation. Recently we identified two enzymes that are involved in the phosphorylation (Pkp1p) and dephosphorylation (Ppp1p) of Pda1p, the alpha-subunit of the pyruvate dehydrogenase complex. Here we provide evidence that two additional mitochondrial proteins, Pkp2p (Ygl059wp) and Ppp2p (Ycr079wp), are engaged in the regulation of this complex by affecting the phosphorylation state of Pda1p. Our data indicate complementary activities of the kinases and a redundant function for the phosphatases. Both proteins are associated with the complex. We propose a model for the role of the regulatory enzymes and the phosphorylation state of Pda1p in the assembly process of the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex of Saccharomyces cerevisiae is regulated by phosphorylation

Mitochondria were isolated from Saccharomyces cerevisiae grown on different carbon sources prior to incubation with [ γ- 32P]ATP. A major 46,000-M r phosphoprotein, corresponding in M r value to the El α subunit of the yeast pyruvate dehydrogenase complex (PDC), was detected only in mitochondria isolated from cells grown on a fermentable carbon source such as galactose. Immunoprecipitation with subunit-specific antiserum to the E1 component of mammalian or yeast PDC confirmed the identity of this polypeptide. PDC activity in isolated yeast mitochondria could be inactivated in an ATP-dependent fashion and reactivated in the presence of Ca 2+ ions.

Enzyme inhibitory autoantibodies to pyruvate dehydrogenase complex in primary biliary cirrhosis differ for mammalian, yeast and bacterial enzymes: Implications for molecular mimicry

Primary biliary cirrhosis is a chronic autoimmune disease in which serum autoantibodies against the mitochondrial 2-oxo acid dehydrogenase enzyme complexes (M2 antibodies) are regularly present. Molecular mimicry of host proteins by bacterial counterparts is a suggested explanation for the origin of these autoantibodies. We tested this hypothesis by measuring the functional reactivity of serum autoantibodies by means of an enzyme inhibition assay against pyruvate dehydrogenase complex from different sources: mammalian, Saccharomyces cerevisiae and Escherichia coli. The 10 primary biliary cirrhosis sera all reacted on immunofluorescence study for M2 antibodies and on immunoblotting with the pyruvate dehydrogenase complex E2 subunit from each of the three enzymes, but there were strikingly different inhibitory capacities. The primary biliary cirrhosis sera were highly inhibitory for mammalian pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level of inhibition, 99%), moderately inhibitory for yeast pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level, 70%) and weakly inhibitory for Escherichia coli pyruvate dehydrogenase complex (4 of 10 inhibitory; mean level, 26%). Thus, with a functional assay that depends on epitope recognition of primary biliary cirrhosis sera, cross-reactivity between mammalian and bacterial pyruvate dehydrogenase complex enzymes is low and molecular mimicry, at least at the B-lymphocyte level, is not supported. (HEPATOLOGY 1994; 19:1029–1033.)

Enzyme inhibitory autoantibodies to pyruvate dehydrogenase complex in primary biliary cirrhosis differ for mammalian, yeast and bacterial enzymes: Implications for molecular mimicry

Primary biliary cirrhosis is a chronic autoimmune disease in which serum autoantibodies against the mitochondrial 2-oxo acid dehydrogenase enzyme complexes (M2 antibodies) are regularly present. Molecular mimicry of host proteins by bacterial counterparts is a suggested explanation for the origin of these autoantibodies. We tested this hypothesis by measuring the functional reactivity of serum autoantibodies by means of an enzyme inhibition assay against pyruvate dehydrogenase complex from different sources: mammalian, Saccharomyces cerevisiae and Escherichia coli. The 10 primary biliary cirrhosis sera all reacted on immunofluorescence study for M2 antibodies and on immunoblotting with the pyruvate dehydrogenase complex E2 subunit from each of the three enzymes, but there were strikingly different inhibitory capacities. The primary biliary cirrhosis sera were highly inhibitory for mammalian pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level of inhibition, 99%), moderately inhibitory for yeast pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level, 70%) and weakly inhibitory for Escherichia coli pyruvate dehydrogenase complex (4 of 10 inhibitory; mean level, 26%). Thus, with a functional assay that depends on epitope recognition of primary biliary cirrhosis sera, cross-reactivity between mammalian and bacterial pyruvate dehydrogenase complex enzymes is low and molecular mimicry, at least at the B-lymphocyte level, is not supported.

Molecular cloning of the gene for the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae

The E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector lambda gt11. The gene encoding the E1 alpha subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V. The identify of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-E1 alpha serum. (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-E1 alpha serum in haploid cells. In addition, the 1.7-kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N-terminal and central regions of the human E1 alpha peptide. We propose to call the gene encoding the E1 alpha subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the lambda gt11 library using the anti-E1 beta serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.

Blink rates and disorders of movement.

Blink rate, a putative noninvasive marker of central dopamine activity, was assessed in medication-free patients with Parkinson's disease, progressive supranuclear palsy, Huntington's disease, and dystonia. The normal control rate of 24 blinks per minute was significantly higher than the rate of 12 and 4 blinks per minute recorded for patients with Parkinson's disease and progressive supranuclear palsy, respectively. The rates for patients with Huntington's disease and dystonia did not differ significantly from those of controls (36 and 26 blinks per minute, respectively).

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Pyruvate dehydrogenase complex from baker's yeast. 1. Purification and some kinetic and regulatory properties.

Pyruvate dehydrogenase complex, for the first time, was highly purified from commercial baker's yeast (saccharomyces cerevisiae). Proteolytic degradation was prevented by the inclusion of the protease inhibitors pepstatin A, leupeptin, and phenylmethanesulfonyl fluoride during the enzyme purification. The yield from 1 kg of pressed yeast was about 15--20 mg enzyme with a specific activity of 17--30 U/mg. Most of the kinetic and regulatory properties of the yeast enzyme were found similar to those of the mammalian mitochondrial pyruvate dehydrogenase complexes except that Km for pyruvate, when assayed at the pH optimum, was much higher than in the mammalian complexes and resembled the values reported for the complexes of gram-negative bacteria. Furthermore, neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylation) as occurs in mammals, plants, and Neurospora crassa.