Ion movements during energy-linked mitochondrial structural changes

Webster, Keith A.; Bronk, J. R.

doi:10.1007/bf00743225

Cited by 20 publications

(6 citation statements)

References 49 publications

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“…The mass of mt-11S is identical to that of purified H6 11S, suggesting efficient localization to the matrix, where the MTS is removed. Under standard culture conditions, 1% galactose resulted in 1.34 ± 0.05 μM of mature 11S in mitochondria, assuming a standard matrix volume of 1.1 μL/mg of protein [19] and using pure H6 11S as a standard (Fig. S5b, c).…”

Section: Resultsmentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes

Pereira

Allen

Watkins

et al. 2019

Journal of Molecular Biology

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Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.

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Section: Resultsmentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes

Pereira

Allen

Watkins

et al. 2019

Journal of Molecular Biology

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show abstract

“…The mass of mt-11S is identical to that of purified H6 11S, suggesting efficient localisation to the matrix, where the MTS is removed. Under standard culture conditions, 1% galactose resulted in 1.34 ± 0.05 µM of mature 11S in mitochondria, assuming a standard matrix volume of 1.1 µL/mg of protein 17 and using pure H6 11S as a standard ( Fig. S3c).…”

Section: Real-time Import Assay In Isolated Yeast Mitochondriamentioning

confidence: 99%

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Pereira

Allen

Watkins

et al. 2018

Preprint

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Abbreviations:AA -antimycin A; IMM -inner mitochondrial membrane; mt-11S -mitochondrial-targeted 11S; H6 11S -Nterminal 6xHis tagged version of 11S; OMM -outer mitochondrial membrane; OVA -oligomycin, valinomycin and antimycin A (full depolarisation cocktail); MTS -mitochondrial targeting sequence, NanoBiT -NanoLuc Binary Technology; PAM -presequence-translocase-associated import-motor; pep86high-affinity version of NanoBiT small fragment; pep114 -small fragment of NanoBiT; PMF -proton motive force; TIM -translocase of the inner membrane; TOM -translocase of the outer membrane; 11Slarge fragment of the NanoBiT; Δ Ψ -membrane potential. 2 ABSTRACTProtein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises due the poor sensitivity, low time-resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes, and allows continuous acquisition with a time-resolution in the order of seconds -yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system, and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigour unattainable through classical methods.

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“…Oxygen consumption was measured using a Clark oxygen electrode as described previously (44). Cells (8ϫ10 6 myo-cytes) were added to 500 L of culture media in a micro water-jacketed cell with magnetic stirrer (Hansatech Instruments Limited, Norfolk, UK).…”

Section: Respiration Measurementsmentioning

confidence: 99%

Mitochondrial signals initiate the activation of c‐Jun N‐terminal kinase (JNK) by hypoxia‐reoxygenation

Dougherty¹,

Kubasiak²,

Frazier³

et al. 2004

The FASEB Journal

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C-Jun N-terminal kinase (JNK) is part of the mitogen-activated protein kinase (MAPK) family of signaling pathways that are induced in response to extracellular stimuli. JNK is primarily a stress-response pathway and can be activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through non-receptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). The molecular initiators of MAPKs by non-receptor stimuli have not been described. Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We show here that the activation of JNK by this condition is initiated in the mitochondria and requires coupled electron transport, ROI generation, and calcium flux. These signals cause the selective, sequential activation of the calcium-dependent, proline-rich kinase Pyk2 and the small GTP binding factors Rac-1 and Cdc42. Interruption of these interactions with inactivated dominant negative mutant proteins, blocking calcium flux, or inhibiting electron transport through mitochondrial complexes II, III, or IV prevents JNK activation and results in a proapoptotic phenotype that is characteristic of JNK inhibition in this model of ischemia-reperfusion. The signaling pathway is unique for the reoxygenation stimulus and provides a framework for other non-receptor-mediated pathways of MAPK activation.

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Ion movements during energy-linked mitochondrial structural changes

Cited by 20 publications

References 49 publications

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes

Mitochondrial signals initiate the activation of c‐Jun N‐terminal kinase (JNK) by hypoxia‐reoxygenation

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