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A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria.

Hoppins S, Collins SR, Cassidy-Stone A, Hummel E, Devay RM, Lackner LL, Westermann B, Schuldiner M, Weissman JS, Nunnari J - J. Cell Biol. (2011)

Bottom Line: The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components.We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology.We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.

ABSTRACT
To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

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MitOS interacts with both outer and inner mitochondrial membranes. (A) Proteomic analysis of FLAG-tag immunoprecipitations as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag purification or untagged wild type control (top row), the number of peptides (and percent coverage) are shown for each identified protein (left column). Data are represented as the mean ± standard error of three independent experiments. Asterisks indicate data represented as the mean ± standard deviation of two independent experiments. (B) Interdependent protein stability of components of the MitOS complex. The indicated relative amounts of whole-cell extract prepared from the indicated MitOS component FLAG-tag strain were subjected to SDS-PAGE and immunoblotting with α-FLAG, α-Ugo1, and α-G6PDH. (C) Genetic connection scatter plot of the average of MitOS component genes AIM5, AIM13, FCJ1, and AIM37. The x axis represents the cosine correlation between the mean of MitOS genes interaction scores, and the y axis indicates the mean interaction score between MitOS genes and each gene in the MITO-MAP. Every point in the scatter plot represents one gene. The cosine correlation values for points corresponding to the selected genes themselves were computed using the mean of the interaction score vectors for the remaining selected genes. In cases where the genetic interaction score was not measured, the point is plotted in gray along the line y = 0. (D) Genetic interaction scatter plot of the average of genes encoding components of MitOS (x axis) and POR1 (y axis). The x axis represents the mean of the genetic interaction scores of AIM5, AIM13, FCJ1, and AIM37 with each gene in the MITO-MAP, and the y axis indicates the mean interaction score of POR1 and each gene in the MITO-MAP. Significant common negative genetic interactions are highlighted. (E) Proteomic analysis of FLAG-tag immune purifications from cross-linked mitochondria as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag protein or untagged wild-type control (top row), the number of peptides and percent coverage are shown for each identified protein (left column). Data are expressed as the mean ± standard error of three independent experiments.
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fig3: MitOS interacts with both outer and inner mitochondrial membranes. (A) Proteomic analysis of FLAG-tag immunoprecipitations as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag purification or untagged wild type control (top row), the number of peptides (and percent coverage) are shown for each identified protein (left column). Data are represented as the mean ± standard error of three independent experiments. Asterisks indicate data represented as the mean ± standard deviation of two independent experiments. (B) Interdependent protein stability of components of the MitOS complex. The indicated relative amounts of whole-cell extract prepared from the indicated MitOS component FLAG-tag strain were subjected to SDS-PAGE and immunoblotting with α-FLAG, α-Ugo1, and α-G6PDH. (C) Genetic connection scatter plot of the average of MitOS component genes AIM5, AIM13, FCJ1, and AIM37. The x axis represents the cosine correlation between the mean of MitOS genes interaction scores, and the y axis indicates the mean interaction score between MitOS genes and each gene in the MITO-MAP. Every point in the scatter plot represents one gene. The cosine correlation values for points corresponding to the selected genes themselves were computed using the mean of the interaction score vectors for the remaining selected genes. In cases where the genetic interaction score was not measured, the point is plotted in gray along the line y = 0. (D) Genetic interaction scatter plot of the average of genes encoding components of MitOS (x axis) and POR1 (y axis). The x axis represents the mean of the genetic interaction scores of AIM5, AIM13, FCJ1, and AIM37 with each gene in the MITO-MAP, and the y axis indicates the mean interaction score of POR1 and each gene in the MITO-MAP. Significant common negative genetic interactions are highlighted. (E) Proteomic analysis of FLAG-tag immune purifications from cross-linked mitochondria as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag protein or untagged wild-type control (top row), the number of peptides and percent coverage are shown for each identified protein (left column). Data are expressed as the mean ± standard error of three independent experiments.

Mentions: One robust cluster in the MITO-MAP contained FCJ1 and three poorly characterized genes, previously identified using a computational strategy to uncover genes with altered inheritance of mitochondria: AIM5, AIM13, and AIM37 (Hess et al., 2009). To determine if these proteins physically interact, we constructed strains harboring functional chromosomal C-terminal FLAG-tagged FCJ1 cluster genes, and purified the tagged proteins from crude digitonin-solubilized mitochondrial extracts. We identified interacting proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by analysis of the number of unique peptides and the percent coverage of detected proteins (Fig. 3 A). As a control, we performed the identical analysis using a wild-type untagged strain. In the purification of Fcj1-FLAG, we identified Aim5 and Aim13, but not Aim37. However, purifications of Aim5-FLAG, Aim13-FLAG, and Aim37-FLAG all contained significant peptides/coverage of each other and Fcj1. From these data, we conclude that Fcj1, Aim5, Aim13, and Aim37 physically interact, as suggested by their genetic interaction profiles in the MITO-MAP.


A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria.

Hoppins S, Collins SR, Cassidy-Stone A, Hummel E, Devay RM, Lackner LL, Westermann B, Schuldiner M, Weissman JS, Nunnari J - J. Cell Biol. (2011)

MitOS interacts with both outer and inner mitochondrial membranes. (A) Proteomic analysis of FLAG-tag immunoprecipitations as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag purification or untagged wild type control (top row), the number of peptides (and percent coverage) are shown for each identified protein (left column). Data are represented as the mean ± standard error of three independent experiments. Asterisks indicate data represented as the mean ± standard deviation of two independent experiments. (B) Interdependent protein stability of components of the MitOS complex. The indicated relative amounts of whole-cell extract prepared from the indicated MitOS component FLAG-tag strain were subjected to SDS-PAGE and immunoblotting with α-FLAG, α-Ugo1, and α-G6PDH. (C) Genetic connection scatter plot of the average of MitOS component genes AIM5, AIM13, FCJ1, and AIM37. The x axis represents the cosine correlation between the mean of MitOS genes interaction scores, and the y axis indicates the mean interaction score between MitOS genes and each gene in the MITO-MAP. Every point in the scatter plot represents one gene. The cosine correlation values for points corresponding to the selected genes themselves were computed using the mean of the interaction score vectors for the remaining selected genes. In cases where the genetic interaction score was not measured, the point is plotted in gray along the line y = 0. (D) Genetic interaction scatter plot of the average of genes encoding components of MitOS (x axis) and POR1 (y axis). The x axis represents the mean of the genetic interaction scores of AIM5, AIM13, FCJ1, and AIM37 with each gene in the MITO-MAP, and the y axis indicates the mean interaction score of POR1 and each gene in the MITO-MAP. Significant common negative genetic interactions are highlighted. (E) Proteomic analysis of FLAG-tag immune purifications from cross-linked mitochondria as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag protein or untagged wild-type control (top row), the number of peptides and percent coverage are shown for each identified protein (left column). Data are expressed as the mean ± standard error of three independent experiments.
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fig3: MitOS interacts with both outer and inner mitochondrial membranes. (A) Proteomic analysis of FLAG-tag immunoprecipitations as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag purification or untagged wild type control (top row), the number of peptides (and percent coverage) are shown for each identified protein (left column). Data are represented as the mean ± standard error of three independent experiments. Asterisks indicate data represented as the mean ± standard deviation of two independent experiments. (B) Interdependent protein stability of components of the MitOS complex. The indicated relative amounts of whole-cell extract prepared from the indicated MitOS component FLAG-tag strain were subjected to SDS-PAGE and immunoblotting with α-FLAG, α-Ugo1, and α-G6PDH. (C) Genetic connection scatter plot of the average of MitOS component genes AIM5, AIM13, FCJ1, and AIM37. The x axis represents the cosine correlation between the mean of MitOS genes interaction scores, and the y axis indicates the mean interaction score between MitOS genes and each gene in the MITO-MAP. Every point in the scatter plot represents one gene. The cosine correlation values for points corresponding to the selected genes themselves were computed using the mean of the interaction score vectors for the remaining selected genes. In cases where the genetic interaction score was not measured, the point is plotted in gray along the line y = 0. (D) Genetic interaction scatter plot of the average of genes encoding components of MitOS (x axis) and POR1 (y axis). The x axis represents the mean of the genetic interaction scores of AIM5, AIM13, FCJ1, and AIM37 with each gene in the MITO-MAP, and the y axis indicates the mean interaction score of POR1 and each gene in the MITO-MAP. Significant common negative genetic interactions are highlighted. (E) Proteomic analysis of FLAG-tag immune purifications from cross-linked mitochondria as described in Materials and methods. For each on-bead digest of the indicated FLAG-tag protein or untagged wild-type control (top row), the number of peptides and percent coverage are shown for each identified protein (left column). Data are expressed as the mean ± standard error of three independent experiments.
Mentions: One robust cluster in the MITO-MAP contained FCJ1 and three poorly characterized genes, previously identified using a computational strategy to uncover genes with altered inheritance of mitochondria: AIM5, AIM13, and AIM37 (Hess et al., 2009). To determine if these proteins physically interact, we constructed strains harboring functional chromosomal C-terminal FLAG-tagged FCJ1 cluster genes, and purified the tagged proteins from crude digitonin-solubilized mitochondrial extracts. We identified interacting proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by analysis of the number of unique peptides and the percent coverage of detected proteins (Fig. 3 A). As a control, we performed the identical analysis using a wild-type untagged strain. In the purification of Fcj1-FLAG, we identified Aim5 and Aim13, but not Aim37. However, purifications of Aim5-FLAG, Aim13-FLAG, and Aim37-FLAG all contained significant peptides/coverage of each other and Fcj1. From these data, we conclude that Fcj1, Aim5, Aim13, and Aim37 physically interact, as suggested by their genetic interaction profiles in the MITO-MAP.

Bottom Line: The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components.We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology.We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.

ABSTRACT
To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

Show MeSH
Related in: MedlinePlus