Limits...
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.

Show MeSH
MitOS forms a complex extended scaffold-like structure on the mitochondrial inner membrane. (A) Cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the inset. (B) Cells expressing Fcj1-mCherry and Aim5-yeGFP were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the insets. Arrows indicate Fcj1-mCherry puncta. Double arrowheads indicate areas labeled exclusively by Aim5-yeGFP. (C) Rho0 cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. Boxes indicate the areas shown in the inset. Bar, 2 µm. (D) Schematic representation of MitOS localization in mitochondria and its role as an organizer of inner membrane structure. BR, boundary membrane region; CJ, cristae junction; MIM, inner mitochondrial membrane; MOM, outer mitochondrial membrane. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3198156&req=5

fig7: MitOS forms a complex extended scaffold-like structure on the mitochondrial inner membrane. (A) Cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the inset. (B) Cells expressing Fcj1-mCherry and Aim5-yeGFP were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the insets. Arrows indicate Fcj1-mCherry puncta. Double arrowheads indicate areas labeled exclusively by Aim5-yeGFP. (C) Rho0 cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. Boxes indicate the areas shown in the inset. Bar, 2 µm. (D) Schematic representation of MitOS localization in mitochondria and its role as an organizer of inner membrane structure. BR, boundary membrane region; CJ, cristae junction; MIM, inner mitochondrial membrane; MOM, outer mitochondrial membrane. Bars, 2 µm.

Mentions: Our data point to MitOS as a major determinant of inner membrane structure and organization. To gain insight into MitOS mechanism, we examined MitOS organization and structure in cells expressing C-terminal MitOS GFP tags. Examination of mitochondrial morphology indicated that among the six MitOS components, only Mos1-GFP was nonfunctional. To delineate the mitochondrial inner membrane, matrix-targeted dsRed (mito-dsRed) was coexpressed and, as expected, labeled relatively uniform mitochondrial tubular structures in cells (Fig. 7 A). In striking contrast, a nonuniform labeling pattern for MitOS-GFP components was observed. Specifically, Fcj1-GFP, Aim13-GFP, and Aim37-GFP formed both discrete punctate and filamentous structures around the inner membrane. In contrast, Aim5-GFP and Mos2-GFP formed filamentous structures that in comparison did not have distinct punctate features (Fig. 7 A). These differences observed in MitOS labeling patterns are consistent with our biochemical and genetic data, which indicate a suborganization within the MitOS architecture (Figs. 3 B and S5 A). To test this idea further, colocalization experiments were performed using cells coexpressing two MitOS components from their chromosomal loci: Fcj1-mCherry and Aim5-GFP, which were chosen because they label apparently distinct punctate and filamentous features of MitOS, respectively. As shown in Fig. 7 B, we observed that Fcj1-mCherry and Aim5-GFP colocalize as expected from proteomic and Western analyses; however, consistent with single label experiments, in many cases we did not observe an enrichment of Aim5-GFP in Fcj1-mCherry–labeled puncta (Fig. 7 B, arrows). Conversely, we observed filamentous regions labeled by Aim5-GFP lacking Fcj1-mCherry signal (Fig. 7 B, double arrowheads). Similar patterns of localization were observed in cells coexpressing chromosomal Fcj1-GFP and Aim5-mCherry, which indicates that the observed labeling patterns were not specific to the fluorescent protein tag (Fig. S5 B). Thus, our data indicate that MitOS components do not form a simple homogenous structure; rather, they interact to build a heterogeneous extended structure within mitochondria.


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 forms a complex extended scaffold-like structure on the mitochondrial inner membrane. (A) Cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the inset. (B) Cells expressing Fcj1-mCherry and Aim5-yeGFP were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the insets. Arrows indicate Fcj1-mCherry puncta. Double arrowheads indicate areas labeled exclusively by Aim5-yeGFP. (C) Rho0 cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. Boxes indicate the areas shown in the inset. Bar, 2 µm. (D) Schematic representation of MitOS localization in mitochondria and its role as an organizer of inner membrane structure. BR, boundary membrane region; CJ, cristae junction; MIM, inner mitochondrial membrane; MOM, outer mitochondrial membrane. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198156&req=5

fig7: MitOS forms a complex extended scaffold-like structure on the mitochondrial inner membrane. (A) Cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the inset. (B) Cells expressing Fcj1-mCherry and Aim5-yeGFP were visualized by light microscopy. Representative images are shown. The boxes indicate the areas shown in the insets. Arrows indicate Fcj1-mCherry puncta. Double arrowheads indicate areas labeled exclusively by Aim5-yeGFP. (C) Rho0 cells expressing GFP-tagged versions of MitOS components as indicated and mito-dsRed were visualized by light microscopy. Representative images are shown. Boxes indicate the areas shown in the inset. Bar, 2 µm. (D) Schematic representation of MitOS localization in mitochondria and its role as an organizer of inner membrane structure. BR, boundary membrane region; CJ, cristae junction; MIM, inner mitochondrial membrane; MOM, outer mitochondrial membrane. Bars, 2 µm.
Mentions: Our data point to MitOS as a major determinant of inner membrane structure and organization. To gain insight into MitOS mechanism, we examined MitOS organization and structure in cells expressing C-terminal MitOS GFP tags. Examination of mitochondrial morphology indicated that among the six MitOS components, only Mos1-GFP was nonfunctional. To delineate the mitochondrial inner membrane, matrix-targeted dsRed (mito-dsRed) was coexpressed and, as expected, labeled relatively uniform mitochondrial tubular structures in cells (Fig. 7 A). In striking contrast, a nonuniform labeling pattern for MitOS-GFP components was observed. Specifically, Fcj1-GFP, Aim13-GFP, and Aim37-GFP formed both discrete punctate and filamentous structures around the inner membrane. In contrast, Aim5-GFP and Mos2-GFP formed filamentous structures that in comparison did not have distinct punctate features (Fig. 7 A). These differences observed in MitOS labeling patterns are consistent with our biochemical and genetic data, which indicate a suborganization within the MitOS architecture (Figs. 3 B and S5 A). To test this idea further, colocalization experiments were performed using cells coexpressing two MitOS components from their chromosomal loci: Fcj1-mCherry and Aim5-GFP, which were chosen because they label apparently distinct punctate and filamentous features of MitOS, respectively. As shown in Fig. 7 B, we observed that Fcj1-mCherry and Aim5-GFP colocalize as expected from proteomic and Western analyses; however, consistent with single label experiments, in many cases we did not observe an enrichment of Aim5-GFP in Fcj1-mCherry–labeled puncta (Fig. 7 B, arrows). Conversely, we observed filamentous regions labeled by Aim5-GFP lacking Fcj1-mCherry signal (Fig. 7 B, double arrowheads). Similar patterns of localization were observed in cells coexpressing chromosomal Fcj1-GFP and Aim5-mCherry, which indicates that the observed labeling patterns were not specific to the fluorescent protein tag (Fig. S5 B). Thus, our data indicate that MitOS components do not form a simple homogenous structure; rather, they interact to build a heterogeneous extended structure within mitochondria.

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