High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes

Wittig, Ilka; Karas, Michael; Schägger, Hermann

doi:10.1074/mcp.m700076-mcp200

Cited by 505 publications

(487 citation statements)

References 37 publications

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“…CN-PAGE was conducted using a 4 to 16% gradient gel according to the protocol described (Wittig et al, 2007). Gels were poured using 16 3 20-cm plates separated with 10-mm spacers.…”

Section: Cn-pagementioning

confidence: 99%

Lateral Segregation of Photosystem I in Cyanobacterial Thylakoids

et al. 2017

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Photosystem I (PSI) is the dominant photosystem in cyanobacteria and it plays a pivotal role in cyanobacterial metabolism. Despite its biological importance, the native organization of PSI in cyanobacterial thylakoid membranes is poorly understood. Here, we use atomic force microscopy (AFM) to show that ordered, extensive macromolecular arrays of PSI complexes are present in thylakoids from Thermosynechococcus elongatus, Synechococcus sp PCC 7002, and Synechocystis sp PCC 6803. Hyperspectral confocal fluorescence microscopy and three-dimensional structured illumination microscopy of Synechocystis sp PCC 6803 cells visualize PSI domains within the context of the complete thylakoid system. Crystallographic and AFM data were used to build a structural model of a membrane landscape comprising 96 PSI trimers and 27,648 chlorophyll a molecules. Rather than facilitating intertrimer energy transfer, the close associations between PSI primarily maximize packing efficiency; short-range interactions with Complex I and cytochrome b 6 f are excluded from these regions of the membrane, so PSI turnover is sustained by long-distance diffusion of the electron donors at the membrane surface. Elsewhere, PSI-photosystem II contact zones provide sites for docking phycobilisomes and the formation of megacomplexes. PSI-enriched domains in cyanobacteria might foreshadow the partitioning of PSI into stromal lamellae in plants, similarly sustained by long-distance diffusion of electron carriers.

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“…CN-PAGE was conducted using a 4 to 16% gradient gel according to the protocol described (Wittig et al, 2007). Gels were poured using 16 3 20-cm plates separated with 10-mm spacers.…”

Section: Cn-pagementioning

confidence: 99%

Lateral Segregation of Photosystem I in Cyanobacterial Thylakoids

et al. 2017

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“…2 An almost complete absence of muscle CI, CIV and CV in-gel activity was observed (Figure 1a). Moreover, CV sub-complexes were also detected.…”

Section: Resultsmentioning

confidence: 97%

Molecular-genetic characterization and rescue of a TSFM mutation causing childhood-onset ataxia and nonobstructive cardiomyopathy

et al. 2016

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Oxidative phosphorylation dysfunction has been found in many different disorders. This biochemical pathway depends on mitochondrial protein synthesis. Thus, mutations in components of the mitochondrial translation system can be responsible for some of these pathologies. We identified a new homozygous missense mutation in the mitochondrial translation elongation factor Ts gene in a patient suffering from slowly progressive childhood ataxia and hypertrophic cardiomyopathy. Using cell, biochemical and molecular-genetic protocols, we confirm it as the etiologic factor of this phenotype. Moreover, as an important functional confirmation, we rescued the normal molecular phenotype by expression of the wild-type TSFM cDNA in patient's fibroblasts. Different TSFM mutations can produce the same or very different clinical phenotypes, going from abortions to moderately severe presentations. On the other hand, the same TSFM mutation can also produce same or different phenotypes within the same range of presentations, therefore suggesting the involvement of unknown factors.

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“…Samples (∼5 mg protein·mL −1 ) were solubilized by incubation on ice for 20 min in 1% (wt/vol) digitonin (Calbiochem) and centrifuged, and 50-60 μg protein (per lane) were applied to 3-12% Trisglycine native-PAGE gels (Invitrogen) and run according to the manufacturer's instructions. Proteins were visualized using colloidal Coomassie R250, or the gel strips were destained with MilliQ water for in-gel assays performed using 150 μM NADH and 1 mg·mL −1 nitro blue tetrazolium (NBT) for complex I, or 84 mM succinate, 0.2 mM phenazine methosulfate (PMS), 2 mg·mL −1 NBT, and 10 mM NaCN for complex II (18).…”

Section: Methodsmentioning

confidence: 99%

“…Fig. 1 compares blue native PAGE analyses (18) of the bovine heart mitochondria, membranes, and SMP preparations used here, revealing that each preparation contains an equivalent complement of supercomplexes. In-gel activity assays confirmed the presence of complex I (as well as complexes III and IV), but not complex II, in the bands attributed to supercomplexes, consistent with previous reports (4,7,13).…”

Section: Confirmation Of the Presence Of Supercomplexes In Submitochomentioning

confidence: 99%

Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes

Blaza

Serreli

Jones

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

163

126

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In mitochondria, four respiratory-chain complexes drive oxidative phosphorylation by sustaining a proton-motive force across the inner membrane that is used to synthesize ATP. The question of how the densely packed proteins of the inner membrane are organized to optimize structure and function has returned to prominence with the characterization of respiratory-chain supercomplexes. Supercomplexes are increasingly accepted structural entities, but their functional and catalytic advantages are disputed. Notably, substrate "channeling" between the enzymes in supercomplexes has been proposed to confer a kinetic advantage, relative to the rate provided by a freely accessible, common substrate pool. Here, we focus on the mitochondrial ubiquinone/ubiquinol pool. We formulate and test three conceptually simple predictions of the behavior of the mammalian respiratory chain that depend on whether channeling in supercomplexes is kinetically important, and on whether the ubiquinone pool is partitioned between pathways. Our spectroscopic and kinetic experiments demonstrate how the metabolic pathways for NADH and succinate oxidation communicate and catalyze via a single, universally accessible ubiquinone/ubiquinol pool that is not partitioned or channeled. We reevaluate the major piece of contrary evidence from flux control analysis and find that the conclusion of substrate channeling arises from the particular behavior of a single inhibitor; we explain why different inhibitors behave differently and show that a robust flux control analysis provides no evidence for channeling. Finally, we discuss how the formation of respiratory-chain supercomplexes may confer alternative advantages on energy-converting membranes.supercomplex | respirasome | mitochondria | respiratory chain | ubiquinone C ontemporary models for the organization of respiratorychain complexes in energy-transducing membranes range from the fluid model to the respirasome model. In the fluid model each complex is a free and independent entity, and substrates exchange freely between enzymes through common ubiquinone/ubiquinol (Q) and cytochrome c ox /c red (cyt c) pools (1-3). In the respirasome model the complexes are assembled into units that contain everything required for catalysis; substrates are channeled between enzymes within the respirasome, not exchanged with external pools (4, 5). Intermediate models, in which some enzymes are free and some assembled into multienzyme supercomplexes, or in which supercomplexes are assembled and disassembled in response to changing conditions, are increasingly considered a realistic combination of these two extremes (6).Structural evidence for respiratory-chain supercomplexes, initiated by observations by blue native PAGE analyses of mammalian mitochondria (4, 7), has culminated in electron microscopy data that revealed the arrangement of complexes I, III, and IV in a particular class of isolated supercomplex assembly (8, 9); respiratory-chain supercomplexes have also been observed (at lower resolution) in the membrane (10...

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High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes

Cited by 505 publications

References 37 publications

Lateral Segregation of Photosystem I in Cyanobacterial Thylakoids

Lateral Segregation of Photosystem I in Cyanobacterial Thylakoids

Molecular-genetic characterization and rescue of a TSFM mutation causing childhood-onset ataxia and nonobstructive cardiomyopathy

Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes

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