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Carla M Koehler - One of the best experts on this subject based on the ideXlab platform.
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a novel Mitochondrial Matrix serine threonine protein phosphatase regulates the mitochondria permeability transition pore and is essential for cellular survival and development
Genes & Development, 2007Co-Authors: Shuxun Ren, Carla M Koehler, Paavo Korge, Jayoung Choi, Yuan Dong, James N Weiss, Jaunian Chen, Yibin WangAbstract:Mitochondria play a central role in the regulation of programmed cell death signaling. Here, we report the finding of a Mitochondrial Matrix-targeted protein phosphatase 2C family member (PP2Cm) that regulates Mitochondrial membrane permeability transition pore (MPTP) opening and is essential for cell survival, embryonic development, and cardiac function. PP2Cm is highly conserved among vertebrates, with the highest expression levels detected in the heart and brain. Small hairpin RNA (shRNA)-mediated knockdown of PP2Cm resulted in cell death associated with loss of Mitochondrial membrane potential in cultured cardiac mycoytes and an induction of hepatocyte apoptosis in vivo. PP2Cm-deficient mitochondria showed elevated susceptibility to calcium-induced MPTP opening, whereas Mitochondrial oxidative phosphorylation activities were not affected. Finally, inactivation of PP2Cm in developing zebrafish embryos caused abnormal cardiac and neural development as well as heart failure associated with induced apoptosis. These data suggest that PP2Cm is a novel Mitochondrial protein phosphatase that has a critical function in cell death and survival, and may play a role in regulating the MPTP opening.
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In vitro analysis of yeast Mitochondrial protein import.
Current protocols in cell biology, 2007Co-Authors: Rosemary A Stuart, Carla M KoehlerAbstract:This unit describes methods for importing in vitro-translated or recombinant proteins into isolated yeast mitochondria and for exporting Mitochondrial proteins translated in the yeast Mitochondrial Matrix into the inner Mitochondrial membrane. The methods use mitochondria isolated from yeast cells and Mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate Mitochondrial Matrix translation of proteins and their export.
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Current Protocols in Cell Biology - In Vitro Analysis of Yeast Mitochondrial Protein Import
Current Protocols in Cell Biology, 2007Co-Authors: Rosemary A Stuart, Carla M KoehlerAbstract:This unit describes methods for importing in vitro–translated or recombinant proteins into isolated yeast mitochondria and for exporting Mitochondrial proteins translated in the yeast Mitochondrial Matrix into the inner Mitochondrial membrane. The methods use mitochondria isolated from yeast cells and Mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate Mitochondrial Matrix translation of proteins and their export. Keywords: mitochondria; Saccharomyces cerevisiae; protein translocation; protein import; protein export; translocon; TOM; TIM
Paul A Cobine - One of the best experts on this subject based on the ideXlab platform.
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copper import into the Mitochondrial Matrix in saccharomyces cerevisiae is mediated by pic2 a Mitochondrial carrier family protein
Journal of Biological Chemistry, 2013Co-Authors: Katherine E Vest, Dennis R Winge, Scot C Leary, Paul A CobineAbstract:Saccharomyces cerevisiae must import copper into the Mitochondrial Matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a Mitochondrial carrier family protein capable of importing copper into the Matrix. In vitro transport of the CuL into the Mitochondrial Matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a Matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total Mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria. Background: Copper must enter the Mitochondrial Matrix prior to assembly into cytochrome c oxidase. Results: Pic2 transports Mitochondrial copper in vivo and in vitro. Conclusion: Pic2 mediates copper import into the Mitochondrial Matrix. Significance: We have identified the first Mitochondrial copper importer.
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copper import into the Mitochondrial Matrix in saccharomyces cerevisiae is mediated by pic2 a Mitochondrial carrier family protein
Journal of Biological Chemistry, 2013Co-Authors: Katherine E Vest, Dennis R Winge, Scot C Leary, Paul A CobineAbstract:Abstract Saccharomyces cerevisiae must import copper into the Mitochondrial Matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic, fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a Mitochondrial carrier family protein capable of importing copper into the Matrix. In vitro transport of CuL into the Mitochondrial Matrix is saturable and temperature-dependent. Strains with a deletion of PIC2 grow poorly on copper-deficient, non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a Matrix targeted copper competitor. Mitochondria from pic2∆ cells have lower total Mitochondrial copper and exhibit a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.
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Mitochondrial Matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase.
Journal of Biological Chemistry, 2006Co-Authors: Paul A Cobine, Fabien Pierrel, Megan L Bestwick, Dennis R WingeAbstract:A Mitochondrial Matrix copper ligand (CuL) complex, conserved in mammalian cells, is the likely source of copper for assembly of cytochrome c oxidase (CcO) and superoxide dismutase 1 (Sod1) within the intermembrane space (IMS) in yeast. Targeting the copper-binding proteins human Sod1 and Crs5 to the Mitochondrial Matrix results in growth impairment on non-fermentable medium caused by decreased levels of CcO. This effect is reversed by copper supplementation. Matrix-targeted Crs5 diminished Sod1 protein within the IMS and impaired activity of an inner membrane tethered human Sod1. Copper binding by the Matrix-targeted proteins attenuates levels of the CuL complex without affecting total Mitochondrial copper. These data suggest that attenuation of the Matrix CuL complex via heterologous competitors limits available copper for metallation of CcO and Sod1 within the IMS. The ligand also exists in the cytoplasm in an apparent metal-free state.
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Yeast contain a non-proteinaceous pool of copper in the Mitochondrial Matrix.
The Journal of biological chemistry, 2004Co-Authors: Paul A Cobine, Kevin Rigby, Luis Ojeda, Dennis R WingeAbstract:Abstract The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known Mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of Mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the Matrix as a soluble, anionic, low molecular weight complex. The identity of the Matrix copper ligand is unknown, but the bulk of the Matrix copper fraction is not protein-bound. The Mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in Mitochondrial copper, yet the expansion of this Matrix pool does not induce any respiration defects. The Matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the Mitochondrial Matrix results in suppression of growth defects of sod2Δ cells. However, in the absence of CCS within the Matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.
Dennis R Winge - One of the best experts on this subject based on the ideXlab platform.
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copper import into the Mitochondrial Matrix in saccharomyces cerevisiae is mediated by pic2 a Mitochondrial carrier family protein
Journal of Biological Chemistry, 2013Co-Authors: Katherine E Vest, Dennis R Winge, Scot C Leary, Paul A CobineAbstract:Saccharomyces cerevisiae must import copper into the Mitochondrial Matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a Mitochondrial carrier family protein capable of importing copper into the Matrix. In vitro transport of the CuL into the Mitochondrial Matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a Matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total Mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria. Background: Copper must enter the Mitochondrial Matrix prior to assembly into cytochrome c oxidase. Results: Pic2 transports Mitochondrial copper in vivo and in vitro. Conclusion: Pic2 mediates copper import into the Mitochondrial Matrix. Significance: We have identified the first Mitochondrial copper importer.
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copper import into the Mitochondrial Matrix in saccharomyces cerevisiae is mediated by pic2 a Mitochondrial carrier family protein
Journal of Biological Chemistry, 2013Co-Authors: Katherine E Vest, Dennis R Winge, Scot C Leary, Paul A CobineAbstract:Abstract Saccharomyces cerevisiae must import copper into the Mitochondrial Matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic, fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a Mitochondrial carrier family protein capable of importing copper into the Matrix. In vitro transport of CuL into the Mitochondrial Matrix is saturable and temperature-dependent. Strains with a deletion of PIC2 grow poorly on copper-deficient, non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a Matrix targeted copper competitor. Mitochondria from pic2∆ cells have lower total Mitochondrial copper and exhibit a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.
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Mitochondrial Matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase.
Journal of Biological Chemistry, 2006Co-Authors: Paul A Cobine, Fabien Pierrel, Megan L Bestwick, Dennis R WingeAbstract:A Mitochondrial Matrix copper ligand (CuL) complex, conserved in mammalian cells, is the likely source of copper for assembly of cytochrome c oxidase (CcO) and superoxide dismutase 1 (Sod1) within the intermembrane space (IMS) in yeast. Targeting the copper-binding proteins human Sod1 and Crs5 to the Mitochondrial Matrix results in growth impairment on non-fermentable medium caused by decreased levels of CcO. This effect is reversed by copper supplementation. Matrix-targeted Crs5 diminished Sod1 protein within the IMS and impaired activity of an inner membrane tethered human Sod1. Copper binding by the Matrix-targeted proteins attenuates levels of the CuL complex without affecting total Mitochondrial copper. These data suggest that attenuation of the Matrix CuL complex via heterologous competitors limits available copper for metallation of CcO and Sod1 within the IMS. The ligand also exists in the cytoplasm in an apparent metal-free state.
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Yeast contain a non-proteinaceous pool of copper in the Mitochondrial Matrix.
The Journal of biological chemistry, 2004Co-Authors: Paul A Cobine, Kevin Rigby, Luis Ojeda, Dennis R WingeAbstract:Abstract The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known Mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of Mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the Matrix as a soluble, anionic, low molecular weight complex. The identity of the Matrix copper ligand is unknown, but the bulk of the Matrix copper fraction is not protein-bound. The Mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in Mitochondrial copper, yet the expansion of this Matrix pool does not induce any respiration defects. The Matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the Mitochondrial Matrix results in suppression of growth defects of sod2Δ cells. However, in the absence of CCS within the Matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.
Rosemary A Stuart - One of the best experts on this subject based on the ideXlab platform.
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In vitro analysis of yeast Mitochondrial protein import.
Current protocols in cell biology, 2007Co-Authors: Rosemary A Stuart, Carla M KoehlerAbstract:This unit describes methods for importing in vitro-translated or recombinant proteins into isolated yeast mitochondria and for exporting Mitochondrial proteins translated in the yeast Mitochondrial Matrix into the inner Mitochondrial membrane. The methods use mitochondria isolated from yeast cells and Mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate Mitochondrial Matrix translation of proteins and their export.
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Current Protocols in Cell Biology - In Vitro Analysis of Yeast Mitochondrial Protein Import
Current Protocols in Cell Biology, 2007Co-Authors: Rosemary A Stuart, Carla M KoehlerAbstract:This unit describes methods for importing in vitro–translated or recombinant proteins into isolated yeast mitochondria and for exporting Mitochondrial proteins translated in the yeast Mitochondrial Matrix into the inner Mitochondrial membrane. The methods use mitochondria isolated from yeast cells and Mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate Mitochondrial Matrix translation of proteins and their export. Keywords: mitochondria; Saccharomyces cerevisiae; protein translocation; protein import; protein export; translocon; TOM; TIM
Jennifer S. Lawton - One of the best experts on this subject based on the ideXlab platform.
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Relationship between Mitochondrial Matrix volume and cellular volume in response to stress and the role of ATP-sensitive potassium channel.
Circulation, 2013Co-Authors: Melissa M. Anastacio, Evelyn M. Kanter, Angela D. Keith, Richard B. Schuessler, Carol M. Makepeace, Haixia Zhang, Jennifer S. LawtonAbstract:Cardiac myocytes demonstrate significant swelling and associated reduced contractility in response to stress that is prevented by the ATP-sensitive potassium channel opener, diazoxide (DZX) via an unknown mechanism. One proposed mechanism of cardioprotection is Mitochondrial Matrix swelling. To establish the relationship between Mitochondrial and cellular volume during stress, this study examined the effect of DZX on Mitochondrial volume. Isolated mouse mitochondria were exposed to the following solutions: Tyrode, isolation buffer, cardioplegia (CPG)±DZX±ATP-sensitive potassium channel inhibitor, 5-hydroxydecanoate, and metabolic inhibition (MI) ± DZX ± 5-hydroxydecanoate. Mitochondrial volume was measured. DZX resulted in significant Mitochondrial swelling (P<0.0001 versus Tyrode). MI and CPG resulted in significant Mitochondrial swelling compared with baseline volume. The addition of DZX did not alter the response of Mitochondrial volume to CPG (P=0.912) but increased swelling in response to MI (P=0.036). The addition of 5-hydroxydecanoate to MI + DZX or CPG+DZX significantly reduced Mitochondrial swelling (P<0.003 MI+DZX versus MI + DZX + 5HD; P<0.001 CPG+DZX versus CPG + DZX + 5HD). Both cellular and Mitochondrial volume increased during exposure to MI and CPG. DZX did not alter Mitochondrial volume during CPG; however, it was associated with an increase in Mitochondrial volume during MI. 5-Hydroxydecanoate reduced Mitochondrial volume during exposure to both stresses with DZX, supporting a role for a Mitochondrial ATP-sensitive potassium channel in the mechanism of cardioprotection by DZX.
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relationship between Mitochondrial Matrix volume and cellular volume in response to stress and the role of atp sensitive potassium channel
Circulation, 2013Co-Authors: Melissa M. Anastacio, Evelyn M. Kanter, Angela D. Keith, Richard B. Schuessler, Carol M. Makepeace, Haixia Zhang, Jennifer S. LawtonAbstract:Background—Cardiac myocytes demonstrate significant swelling and associated reduced contractility in response to stress that is prevented by the ATP-sensitive potassium channel opener, diazoxide (DZX) via an unknown mechanism. One proposed mechanism of cardioprotection is Mitochondrial Matrix swelling. To establish the relationship between Mitochondrial and cellular volume during stress, this study examined the effect of DZX on Mitochondrial volume. Methods and Results—Isolated mouse mitochondria were exposed to the following solutions: Tyrode, isolation buffer, cardioplegia (CPG)±DZX±ATP-sensitive potassium channel inhibitor, 5-hydroxydecanoate, and metabolic inhibition (MI)±DZX±5-hydroxydecanoate. Mitochondrial volume was measured. DZX resulted in significant Mitochondrial swelling (P<0.0001 versus Tyrode). MI and CPG resulted in significant Mitochondrial swelling compared with baseline volume. The addition of DZX did not alter the response of Mitochondrial volume to CPG (P=0.912) but increased swelling...