Direct Evidence for Metabolon Formation and Substrate Channeling in Recombinant TCA Cycle Enzymes

Bulutoglu, Beyza; Garcia, Kristen; Wu, Fei; Minteer, Shelley D.; Banta, Scott

doi:10.1021/acschembio.6b00523

Cited by 80 publications

(105 citation statements)

References 48 publications

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“…If respiratory-chain supercomplex assemblies contain the quinone/quinol substrate and channel it between their component enzymes, then a competing quinol oxidase, outside the supercomplex structure, should not be able to turn over (Bulutoglu et al., 2016, Wheeldon et al., 2016) (Figure 1). To test the effects of adding a competing quinol oxidase to mammalian respiratory membranes, we added 0.1 mg AOX per mg SMPs (hereafter written as 0.1 mg AOX mg −1 ) directly to a suspension of SMPs while monitoring the rate of NADH oxidation (NADH:O 2 oxidoreduction).…”

Section: Resultsmentioning

confidence: 99%

“…Electrostatically guided, bounded diffusion (Bauler et al., 2010, Wheeldon et al., 2016) occurs in the malate dehydrogenase/citrate synthase complex of the tricarboxylic acid cycle: the negatively charged oxaloacetate is channeled along a positively charged surface region linking the two active sites (Bulutoglu et al., 2016). In contrast, both quinone and quinol are neutral and extremely hydrophobic molecules, rendering them unsuitable substrates for this mechanism of channeling.…”

Section: Discussionmentioning

confidence: 99%

“…Introduction of an external enzyme to compete for a potentially channeled substrate is a diagnostic test for channeling (Bulutoglu et al., 2016, Wheeldon et al., 2016). If the substrate is truly channeled, flux through the competing pathway is negligible.…”

Section: Introductionmentioning

confidence: 99%

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Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

2018

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SummaryMitochondrial respiratory supercomplexes, comprising complexes I, III, and IV, are the minimal functional units of the electron transport chain. Assembling the individual complexes into supercomplexes may stabilize them, provide greater spatiotemporal control of respiration, or, controversially, confer kinetic advantages through the sequestration of local quinone and cytochrome c pools (substrate channeling). Here, we have incorporated an alternative quinol oxidase (AOX) into mammalian heart mitochondrial membranes to introduce a competing pathway for quinol oxidation and test for channeling. AOX substantially increases the rate of NADH oxidation by O2 without affecting the membrane integrity, the supercomplexes, or NADH-linked oxidative phosphorylation. Therefore, the quinol generated in supercomplexes by complex I is reoxidized more rapidly outside the supercomplex by AOX than inside the supercomplex by complex III. Our results demonstrate that quinone and quinol diffuse freely in and out of supercomplexes: substrate channeling does not occur and is not required to support respiration.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

2018

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“…Examples include protein biosynthesis, RNA transcription, TCA cycle, glycolysis, nucleotide biosynthesis, and even DNA biosynthesis [45–50]. In some cases, protein complexes have been observed in vivo, yet they have been difficult to isolate in vitro possibly due to weak interactions that dissociate during purification [51–53].…”

Section: Discussionmentioning

confidence: 99%

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis

Miao

Lingeman

et al. 2016

PLoS ONE

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We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

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“…Another possibility is that UgtP and/or OpgH utilizes interactions with FtsZ to localize to regions in the cell where UDP-glucose is initially produced and metabolized. There are numerous protein-protein interaction and substrate channeling studies that certainly argue that bacteria are too clever to depend on diffusion alone to control metabolite flow (83)(84)(85)(86)(87)(88)(89)(90). Although the in vitro data argue for possible FtsZ-regulating mechanisms, especially for UgtP, any protein that interacts with an FtsZ surface may conceivably act as an inhibitor of FtsZ activity in vitro.…”

Section: Udp-glucose and Ugtp (B Subtilis)mentioning

confidence: 99%

Metabolism Shapes the Cell

Sperber

Herman

2017

J Bacteriol

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More than 5 decades of work support the idea that cell envelope synthesis, including the inward growth of cell division, is tightly coordinated with DNA replication and protein synthesis through central metabolism. Remarkably, no unifying model exists to account for how these fundamentally disparate processes are functionally coupled. Recent studies demonstrate that proteins involved in carbohydrate and nitrogen metabolism can moonlight as direct regulators of cell division, coordinate cell division and DNA replication, and even suppress defects in DNA replication. In this minireview, we focus on studies illustrating the intimate link between metabolism and regulation of peptidoglycan (PG) synthesis during growth and division, and we identify the following three recurring themes. (i) Nutrient availability, not growth rate, is the primary determinant of cell size. (ii) The degree of gluconeogenic flux is likely to have a profound impact on the metabolites available for cell envelope synthesis, so growth medium selection is a critical consideration when designing and interpreting experiments related to morphogenesis. (iii) Perturbations in pathways relying on commonly shared and limiting metabolites, like undecaprenyl phosphate (Und-P), can lead to pleotropic phenotypes in unrelated pathways.KEYWORDS FtsZ, MreB, UDP-glucose, cell division, gluconeogenesis, metabolism, morphogenesis, peptidoglycan, phosphoenolpyruvate, undecaprenyl phosphate T o accurately partition chromosomes and other cell contents during reproduction, cells must possess mechanisms to organize repeated cycles of cell growth, chromosome replication, and division. Eukaryotes orchestrate this coordination using the cell cycle and separate growth, DNA synthesis, and cytokinesis into distinct, temporally sequestered phases. Bacteria, by contrast, simultaneously increase in cell size and replicate DNA before (or concurrently with) cell division. Elucidating the molecular mechanisms prokaryotes employ to achieve spatiotemporal organization of these intertwined yet functionally disparate processes is of considerable interest to scientists seeking to understand bacterial reproduction, and many outstanding questions remain to be answered. For example, how is DNA replication kept in sync with changing growth and division rates? How are cell dimensions maintained or actively rearranged in response to environmental or developmental cues? What signals do cells sense to switch between increasing in cell size and dividing during the cell cycle? Relatedly, how are these signals transduced to activate/deactivate the distinct machineries required for each process?Perhaps one of the biggest mysteries remaining in bacterial cell biology relates to understanding the regulatory cross talk that must occur to integrate central metabolism with macromolecular biosynthesis. Nutrients are converted into stored energy and precursors used to synthesize macromolecules like DNA and peptidoglycan (PG), so it is no surprise that nutrient availability has a profound impac...

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Direct Evidence for Metabolon Formation and Substrate Channeling in Recombinant TCA Cycle Enzymes

Cited by 80 publications

References 48 publications

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis

Metabolism Shapes the Cell

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