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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 and ATP synthase dimers function in concert to control inner membrane structure. (A) Wild-type and Δfcj1 strains with (rho+) or without (rho0) mtDNA expressing matrix-targeted GFP were visualized by light microscopy (top). Representative images are shown. The graph represents quantification of the mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments characterizing the mitochondrial morphology of ≥75 cells in each replicate. Rho0 wild-type and Δfcj1 strains were analyzed by EM as described (bottom). Representative images are shown. (B) Indicated strains expressing matrix-targeted GFP were grown in SD-dextrose and visualized by light microscopy. Representative images are shown. Bar, 2 µm. The graph represents quantification of the observed mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments (error bars) characterizing the mitochondrial morphology of ≥75 cells in each replicate. (C) Genetic connection scatter plot generated for ATP1, ATP5, and ATP12 as described in Fig. 2 C. Bars: (A, top) 2 µm; (A, bottom) 200 nm; (B) 2 µm.
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fig6: MitOS and ATP synthase dimers function in concert to control inner membrane structure. (A) Wild-type and Δfcj1 strains with (rho+) or without (rho0) mtDNA expressing matrix-targeted GFP were visualized by light microscopy (top). Representative images are shown. The graph represents quantification of the mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments characterizing the mitochondrial morphology of ≥75 cells in each replicate. Rho0 wild-type and Δfcj1 strains were analyzed by EM as described (bottom). Representative images are shown. (B) Indicated strains expressing matrix-targeted GFP were grown in SD-dextrose and visualized by light microscopy. Representative images are shown. Bar, 2 µm. The graph represents quantification of the observed mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments (error bars) characterizing the mitochondrial morphology of ≥75 cells in each replicate. (C) Genetic connection scatter plot generated for ATP1, ATP5, and ATP12 as described in Fig. 2 C. Bars: (A, top) 2 µm; (A, bottom) 200 nm; (B) 2 µm.

Mentions: To determine the role of MitOS in the maintenance of normal mitochondrial ultrastructure, we explored its relationship to ATP synthase by examining mitochondrial morphology and ultrastructure in Δfcj1 cells lacking mtDNA (rho0), which lack assembled respiratory chain complexes. Large lamellar mitochondrial regions observed in Δfcj1 rho+ cells were absent in Δfcj1 rho0 cells, whose mitochondria were indistinguishable from wild-type rho0 control cells (Fig. 6 A, top). Significantly, the abnormally long mitochondrial inner membrane/cristae phenotype observed in Δfcj1 rho+ cells was also suppressed in Δfcj1 cells lacking mtDNA (Fig. 6 A, bottom). Indeed, cristae structures were only rarely detectable in either wild-type or Δfcj1 rho0 cells, which indicates the importance of respiratory complexes for inner membrane structure and cristae biogenesis. We also analyzed double mutant Δfcj1Δcbs1, Δfcj1Δmss51, and Δfcj1Δatp10 cells, which are selectively defective in the assembly of respiratory chain complex III (CIII, cytochrome bc1 complex), complex IV (CIV, cytochrome oxidase complex), or the ATP synthase (CV), respectively (Poutre and Fox, 1987; Costanzo and Fox, 1988; Ackerman and Tzagoloff, 1990; Decoster et al., 1990; Tzagoloff et al., 2004; Herrmann and Funes, 2005). Mitochondria in all three single respiratory chain assembly mutants were disorganized, but were not lamellar in structure (Fig. 6 B, top). In contrast, a large proportion of Δfcj1Δcbs1, Δfcj1Δmss51 cells possessed large, lamellar regions in the mitochondrial network, similar to those observed in Δfcj1 (Fig. 6 B, bottom). In contrast, Δfcj1Δatp10 cells possessed a mitochondrial morphology similar to that observed for Δatp10 cells, which indicates that MitOS-dependent mitochondrial morphology defects specifically require assembled ATP synthase. Further support for this comes from the MITO-MAP, in which we observed a highly correlated ATP synthase cluster consisting of three ATP synthase genes, which encode structural components of ATP synthase required for monomer formation: ATP1, ATP5, and ATP12. Consistent with our analysis, MitOS genes had strong genetic interactions with ATP1, ATP5, and ATP12 (Fig. 6 C). Together, our data suggest a model where the presence of ATP synthase dimers drives the formation of normal cristae membranes in a MitOS-dependent manner.


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 and ATP synthase dimers function in concert to control inner membrane structure. (A) Wild-type and Δfcj1 strains with (rho+) or without (rho0) mtDNA expressing matrix-targeted GFP were visualized by light microscopy (top). Representative images are shown. The graph represents quantification of the mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments characterizing the mitochondrial morphology of ≥75 cells in each replicate. Rho0 wild-type and Δfcj1 strains were analyzed by EM as described (bottom). Representative images are shown. (B) Indicated strains expressing matrix-targeted GFP were grown in SD-dextrose and visualized by light microscopy. Representative images are shown. Bar, 2 µm. The graph represents quantification of the observed mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments (error bars) characterizing the mitochondrial morphology of ≥75 cells in each replicate. (C) Genetic connection scatter plot generated for ATP1, ATP5, and ATP12 as described in Fig. 2 C. Bars: (A, top) 2 µm; (A, bottom) 200 nm; (B) 2 µm.
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fig6: MitOS and ATP synthase dimers function in concert to control inner membrane structure. (A) Wild-type and Δfcj1 strains with (rho+) or without (rho0) mtDNA expressing matrix-targeted GFP were visualized by light microscopy (top). Representative images are shown. The graph represents quantification of the mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments characterizing the mitochondrial morphology of ≥75 cells in each replicate. Rho0 wild-type and Δfcj1 strains were analyzed by EM as described (bottom). Representative images are shown. (B) Indicated strains expressing matrix-targeted GFP were grown in SD-dextrose and visualized by light microscopy. Representative images are shown. Bar, 2 µm. The graph represents quantification of the observed mitochondrial morphology of the indicated strains. Data are represented as the mean ± standard error of three independent experiments (error bars) characterizing the mitochondrial morphology of ≥75 cells in each replicate. (C) Genetic connection scatter plot generated for ATP1, ATP5, and ATP12 as described in Fig. 2 C. Bars: (A, top) 2 µm; (A, bottom) 200 nm; (B) 2 µm.
Mentions: To determine the role of MitOS in the maintenance of normal mitochondrial ultrastructure, we explored its relationship to ATP synthase by examining mitochondrial morphology and ultrastructure in Δfcj1 cells lacking mtDNA (rho0), which lack assembled respiratory chain complexes. Large lamellar mitochondrial regions observed in Δfcj1 rho+ cells were absent in Δfcj1 rho0 cells, whose mitochondria were indistinguishable from wild-type rho0 control cells (Fig. 6 A, top). Significantly, the abnormally long mitochondrial inner membrane/cristae phenotype observed in Δfcj1 rho+ cells was also suppressed in Δfcj1 cells lacking mtDNA (Fig. 6 A, bottom). Indeed, cristae structures were only rarely detectable in either wild-type or Δfcj1 rho0 cells, which indicates the importance of respiratory complexes for inner membrane structure and cristae biogenesis. We also analyzed double mutant Δfcj1Δcbs1, Δfcj1Δmss51, and Δfcj1Δatp10 cells, which are selectively defective in the assembly of respiratory chain complex III (CIII, cytochrome bc1 complex), complex IV (CIV, cytochrome oxidase complex), or the ATP synthase (CV), respectively (Poutre and Fox, 1987; Costanzo and Fox, 1988; Ackerman and Tzagoloff, 1990; Decoster et al., 1990; Tzagoloff et al., 2004; Herrmann and Funes, 2005). Mitochondria in all three single respiratory chain assembly mutants were disorganized, but were not lamellar in structure (Fig. 6 B, top). In contrast, a large proportion of Δfcj1Δcbs1, Δfcj1Δmss51 cells possessed large, lamellar regions in the mitochondrial network, similar to those observed in Δfcj1 (Fig. 6 B, bottom). In contrast, Δfcj1Δatp10 cells possessed a mitochondrial morphology similar to that observed for Δatp10 cells, which indicates that MitOS-dependent mitochondrial morphology defects specifically require assembled ATP synthase. Further support for this comes from the MITO-MAP, in which we observed a highly correlated ATP synthase cluster consisting of three ATP synthase genes, which encode structural components of ATP synthase required for monomer formation: ATP1, ATP5, and ATP12. Consistent with our analysis, MitOS genes had strong genetic interactions with ATP1, ATP5, and ATP12 (Fig. 6 C). Together, our data suggest a model where the presence of ATP synthase dimers drives the formation of normal cristae membranes in a MitOS-dependent manner.

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