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Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability.

Hobbs AE, Srinivasan M, McCaffery JM, Jensen RE - J. Cell Biol. (2001)

Bottom Line: We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids.We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature.Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.

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Mmm1p is localized in punctate structures on the mitochondrial surface. mmm1::URA3 strain YAAH1, which expresses Mmm1p-GFP from plasmid pAA2, was grown in YEP glycerol/ethanol media to early log phase and stained with MitoTracker™ Red. Live cells were mounted on slides and examined using a DeltaVision microscope system. 15 total images were taken in the z axis through the cells, deconvolved, and 7 of the 15 optical sections near the top of the cells were flattened into a single image. (A) Differential interference contrast (DIC); (B) Mmm1p-GFP fluorescence; (C) MitoTracker fluorescence; (D) merged images of B and C. Bar: 2 μm. (E and F) Strain YAAH3, which expresses Mmm1p-HA, was fixed, embedded, and frozen. Cryosections were incubated with antibodies to the HA epitope, followed by incubation with secondary antibodies conjugated to 5-nm gold particles. After staining, sections were examined under the electron microscope. Bar: 0.1 μm.
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Figure 1: Mmm1p is localized in punctate structures on the mitochondrial surface. mmm1::URA3 strain YAAH1, which expresses Mmm1p-GFP from plasmid pAA2, was grown in YEP glycerol/ethanol media to early log phase and stained with MitoTracker™ Red. Live cells were mounted on slides and examined using a DeltaVision microscope system. 15 total images were taken in the z axis through the cells, deconvolved, and 7 of the 15 optical sections near the top of the cells were flattened into a single image. (A) Differential interference contrast (DIC); (B) Mmm1p-GFP fluorescence; (C) MitoTracker fluorescence; (D) merged images of B and C. Bar: 2 μm. (E and F) Strain YAAH3, which expresses Mmm1p-HA, was fixed, embedded, and frozen. Cryosections were incubated with antibodies to the HA epitope, followed by incubation with secondary antibodies conjugated to 5-nm gold particles. After staining, sections were examined under the electron microscope. Bar: 0.1 μm.

Mentions: Previous studies have shown that Mmm1p is a mitochondrial outer membrane protein required to maintain mitochondrial shape. To further examine Mmm1p function, we localized Mmm1p in living yeast cells by expressing the green fluorescent protein fused to the carboxyl terminus of Mmm1p in cells disrupted in MMM1. We found that Mmm1p-GFP rescued the growth defect of mmm1::URA3 cells, demonstrating that the fusion protein is functional, and Western blots confirmed that the fusion protein was intact in yeast cells (A. Aiken Hobbs, unpublished observations). By fluorescence microscopy, the Mmm1p-GFP protein was concentrated in distinct, dot-like structures that colocalized with mitochondrial tubules (Fig. 1). Cells contained on average between five and eight Mmm1p-GFP–containing dots per cell, and 3-D reconstruction of optical slices taken through yeast cells showed that each punctate structure was attached to a mitochondrion (see online movie). While the distribution of dots appeared to be random along the mitochondrial tubule, we noticed that small buds almost always contained an Mmm1p-GFP–containing dot at the bud neck. In contrast to Mmm1p, most other mitochondrial outer membrane proteins, including an OM45p-GFP fusion protein (K. Cerveny, unpublished observations), Tom6p-GFP (Okamoto et al. 1998), and Fzo1p (Hermann et al. 1998), are distributed uniformly along the organelle surface. To confirm this unusual location for Mmm1p, we examined immunogold-labeled sections of cells expressing an Mmm1p-HA fusion protein by electron microscopy (Fig. 1E and Fig. F). Consistent with the localization of Mmm1p-GFP in live cells, Mmm1p-HA was clustered at distinct sites associated with mitochondria.


Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability.

Hobbs AE, Srinivasan M, McCaffery JM, Jensen RE - J. Cell Biol. (2001)

Mmm1p is localized in punctate structures on the mitochondrial surface. mmm1::URA3 strain YAAH1, which expresses Mmm1p-GFP from plasmid pAA2, was grown in YEP glycerol/ethanol media to early log phase and stained with MitoTracker™ Red. Live cells were mounted on slides and examined using a DeltaVision microscope system. 15 total images were taken in the z axis through the cells, deconvolved, and 7 of the 15 optical sections near the top of the cells were flattened into a single image. (A) Differential interference contrast (DIC); (B) Mmm1p-GFP fluorescence; (C) MitoTracker fluorescence; (D) merged images of B and C. Bar: 2 μm. (E and F) Strain YAAH3, which expresses Mmm1p-HA, was fixed, embedded, and frozen. Cryosections were incubated with antibodies to the HA epitope, followed by incubation with secondary antibodies conjugated to 5-nm gold particles. After staining, sections were examined under the electron microscope. Bar: 0.1 μm.
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Related In: Results  -  Collection

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Figure 1: Mmm1p is localized in punctate structures on the mitochondrial surface. mmm1::URA3 strain YAAH1, which expresses Mmm1p-GFP from plasmid pAA2, was grown in YEP glycerol/ethanol media to early log phase and stained with MitoTracker™ Red. Live cells were mounted on slides and examined using a DeltaVision microscope system. 15 total images were taken in the z axis through the cells, deconvolved, and 7 of the 15 optical sections near the top of the cells were flattened into a single image. (A) Differential interference contrast (DIC); (B) Mmm1p-GFP fluorescence; (C) MitoTracker fluorescence; (D) merged images of B and C. Bar: 2 μm. (E and F) Strain YAAH3, which expresses Mmm1p-HA, was fixed, embedded, and frozen. Cryosections were incubated with antibodies to the HA epitope, followed by incubation with secondary antibodies conjugated to 5-nm gold particles. After staining, sections were examined under the electron microscope. Bar: 0.1 μm.
Mentions: Previous studies have shown that Mmm1p is a mitochondrial outer membrane protein required to maintain mitochondrial shape. To further examine Mmm1p function, we localized Mmm1p in living yeast cells by expressing the green fluorescent protein fused to the carboxyl terminus of Mmm1p in cells disrupted in MMM1. We found that Mmm1p-GFP rescued the growth defect of mmm1::URA3 cells, demonstrating that the fusion protein is functional, and Western blots confirmed that the fusion protein was intact in yeast cells (A. Aiken Hobbs, unpublished observations). By fluorescence microscopy, the Mmm1p-GFP protein was concentrated in distinct, dot-like structures that colocalized with mitochondrial tubules (Fig. 1). Cells contained on average between five and eight Mmm1p-GFP–containing dots per cell, and 3-D reconstruction of optical slices taken through yeast cells showed that each punctate structure was attached to a mitochondrion (see online movie). While the distribution of dots appeared to be random along the mitochondrial tubule, we noticed that small buds almost always contained an Mmm1p-GFP–containing dot at the bud neck. In contrast to Mmm1p, most other mitochondrial outer membrane proteins, including an OM45p-GFP fusion protein (K. Cerveny, unpublished observations), Tom6p-GFP (Okamoto et al. 1998), and Fzo1p (Hermann et al. 1998), are distributed uniformly along the organelle surface. To confirm this unusual location for Mmm1p, we examined immunogold-labeled sections of cells expressing an Mmm1p-HA fusion protein by electron microscopy (Fig. 1E and Fig. F). Consistent with the localization of Mmm1p-GFP in live cells, Mmm1p-HA was clustered at distinct sites associated with mitochondria.

Bottom Line: We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids.We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature.Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.

Show MeSH
Related in: MedlinePlus