Monitoring checkpoints of metabolism and protein biogenesis in mitochondria by Phos-tag technology

Walter, Corvin; Marada, Adinarayana; Meisinger, Chris

doi:10.1016/j.jprot.2021.104430

Cited by 7 publications

(3 citation statements)

References 35 publications

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“…Although previous studies, from the 1960s to the present, have focused on the fact that PDKs regulate pyruvate dehydrogenase activity (Linn et al, 1969;Gey et al, 2008;Guo et al, 2017a;Cardoso et al, 2020), our data now demonstrate that the PDKs have additional substrates and that their activity toward these non-PDC substrates can be reciprocally regulated by the PDC itself. This regulation may be mediated via allosteric interactions, as it was previously shown that Pkp1 and Pkp2 function as a heterodimer (Gey et al, 2008;Breitkreutz et al, 2010;Walter et al, 2022) and both Aup1 and Pkp1 stably interact with the PDC (Krause- Buchholz et al, 2006;Guo et al, 2017a). Hundreds of mitochondrial matrix proteins have been documented to undergo phosphorylation (Reinders et al, 2007;Kruse & Højlund, 2017;Renvois é et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation

et al. 2023

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The mitophagic degradation of mitochondrial matrix proteins inSaccharomyces cerevisiaewas 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.

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Section: Discussionmentioning

confidence: 99%

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation

et al. 2023

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“…While previous studies, from the 1960’s to the present, have focused on the fact that PDKs regulate pyruvate dehydrogenase activity (Linn et al ., 1969; Gey et al ., 2008; Guo et al ., 2017a; Cardoso et al ., 2020), our data now demonstrate that the PDKs have additional substrates, and that their activity towards these non-PDC substrates can be reciprocally regulated by the PDC. This regulation may be mediated via allosteric interactions, as it was previously shown that Pkp1 and Pkp2 function as a heterodimer (Gey et al ., 2008; Breitkreutz et al ., 2010; Walter et al ., 2022) and both Aup1 and Pkp1 stably interact with the PDC (Krause-Buchholz et al ., 2006; Guo et al ., 2017a). Hundreds of mitochondrial matrix proteins have been documented to undergo phosphorylation (Reinders et al ., 2007; Kruse and Højlund, 2017; Renvoisé et al ., 2017).…”

Section: Discussionmentioning

confidence: 99%

The pyruvate dehydrogenase complex regulates matrix protein phosphorylation and mitophagic selectivity

Kolitsida

Nolic

Zhou

et al. 2022

Preprint

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The mitophagic degradation of mitochondrial matrix proteins is selective, reflecting a pre-engulfment sorting step. 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 proteins also regulate the phosphorylation and catalytic activity of the yeast pyruvate dehydrogenase complex. To understand the relationship between these two functions, we tested the effect of deleting PDA1 on mitophagic selectivity. We report that pda1Δ cells show a mitophagy selectivity phenotype nearly identical to that of the pkp1Δ pkp2Δ double mutant. However, this is not due to a role for pyruvate dehydrogenase enzymatic activity in regulating mitophagy. Rather, our data suggest a novel mechanism wherein the pyruvate dehydrogenase complex directly regulates its cognate kinases and phosphatases, independent of its catalytic activity, to determine the phosphorylation state of mitochondrial matrix proteins in response to metabolic cues.

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“…In Gram-negative bacteria, PDH consists of 24 catalytic subunits of pyruvate dehydrogenase (E1), 24 catalytic subunits of dihydrolipoyl transacetylase (E2) and 12 catalytic subunits of dihydrolipoate dehydrogenase ([E1] 24 [E2] 24 [E3] 12 ), and has a molecular mass of ~ 4600 kDa 34 , whereas this complex doubles to 8000 ~ 10000 kDa in Gram-positive bacteria and eukaryotes with a representative composition of [E1] 30 [E2] 60 [E3] 12 35 . Besides PDH, the average mass of ACC complex in both prokaryotes and eukaryotes is ~ 265 kDa.…”

Section: Ncm Pathway Circumvents Multiple Inhibitions and Complicated...mentioning

confidence: 99%

A non-carboxylative route for the efficient synthesis of central metabolite malonyl-CoA and its derived products

Tan

et al. 2023

Preprint

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While acetyl-CoA carboxylation is the canonical route for endogenous malonyl-CoA formation found in nature, this pathway suffers from slow kinetics, carbon and energy inefficiencies, tight regulations, complicated architecture and severe cellular toxicity, which limit flux towards malonyl-CoA and become a decisive bottleneck limiting the biosynthesis of all malonyl-CoA-derived products (MDPs). In this study, we built the first non-natural route for malonyl-CoA biosynthesis. The designed non-carboxylative malonyl-CoA (NCM) pathway adopts an “acetyl-CoA-independent” mode, thus avoiding carbon loss, ATP consumption and problematic cross-talk with native metabolic networks. The feasibility of this design was demonstrated by mining and recruiting β-alanine-pyruvate transaminase and malonyl-CoA reductase. The NCM catalysis displays a fast kinetics, with the rate being 103-fold higher than that of natural pathway, representing the fastest malonyl-CoA formation rate to the best of our knowledge. Moreover, the NCM catalysis circumvents both multiple tight regulations and extremely complicated architecture associated with natural pathway, with the mass of comprising enzymes reducing by 93%~97%. In addition, the NCM pathway can substitute natural pathway for malonyl-CoA formation within cells, and does not lead to severe cellular toxicity, but rather improved cell’s robustness to a broad range of challenges. Furthermore, introduction of this pathway into multiple microbes including Escherichia coli, Streptomyces gilvosporeus and Saccharopolyspora spinosa greatly improved production of a variety of highly valuable MDPs including short-chain fatty acid and representative phenol, quinone, alkene, aminoglycoside and macrolide polyketide families, with the production of spinosad in S. spinosa reaching an unprecedented level. In summary, this new-to-nature malonyl-CoA formation pathway circumvents nearly all intrinsic inefficiencies of natural pathway, and can serve as a novel and versatile platform for targeting a repertoire of MDPs with applications as fuels, fine chemicals and pharmaceuticals.

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Monitoring checkpoints of metabolism and protein biogenesis in mitochondria by Phos-tag technology

Cited by 7 publications

References 35 publications

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation

The pyruvate dehydrogenase complex regulates matrix protein phosphorylation and mitophagic selectivity

A non-carboxylative route for the efficient synthesis of central metabolite malonyl-CoA and its derived products

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