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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 maintains a punctate localization in the absence of actin or mtDNA. (A) YAAH1, which expresses Mmm1p-GFP, was grown to early log phase and treated with Latrunculin A (+LatA), or mock treated (−LatA) at 24°C for 30 min. Cells were fixed and stained with Rhodamine-Phalloidin to visualize the actin cytoskeleton. Representative images of cells examined by fluorescence microscopy in the green (GFP) and red (Rh.-Phalloidin) channels are shown. (B) Mmm1p-GFP–expressing strains YAAH1, which contains mtDNA (ρ+) and YAAH2, which lacks mtDNA (ρ°), were stained with 1 μg/ml DAPI to visualize the mtDNA. Although DAPI treatment of living cells preferentially stains mtDNA, low-level staining of nuclear DNA can be seen when images are overexposed or no mtDNA is present. Bars: 2 μm.
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Figure 3: Mmm1p maintains a punctate localization in the absence of actin or mtDNA. (A) YAAH1, which expresses Mmm1p-GFP, was grown to early log phase and treated with Latrunculin A (+LatA), or mock treated (−LatA) at 24°C for 30 min. Cells were fixed and stained with Rhodamine-Phalloidin to visualize the actin cytoskeleton. Representative images of cells examined by fluorescence microscopy in the green (GFP) and red (Rh.-Phalloidin) channels are shown. (B) Mmm1p-GFP–expressing strains YAAH1, which contains mtDNA (ρ+) and YAAH2, which lacks mtDNA (ρ°), were stained with 1 μg/ml DAPI to visualize the mtDNA. Although DAPI treatment of living cells preferentially stains mtDNA, low-level staining of nuclear DNA can be seen when images are overexposed or no mtDNA is present. Bars: 2 μm.

Mentions: Yeast mitochondria have been shown to interact with actin filaments (Drubin et al. 1993; Smith et al. 1995) and Mmm1p appears to be important for this interaction (Boldogh et al. 1998). Nevertheless, an intact actin cytoskeleton was not required for Mmm1p distribution. Mmm1p-GFP–containing cells were treated with Latrunculin A to disrupt the actin cytoskeleton (Ayscough et al. 1997) and examined by fluorescence microscopy (Fig. 3 A). Under conditions where normal actin cables and patches cannot be seen by rhodamine-phalloidin staining, the same number of Mmm1p-GFP dots were still present.


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 maintains a punctate localization in the absence of actin or mtDNA. (A) YAAH1, which expresses Mmm1p-GFP, was grown to early log phase and treated with Latrunculin A (+LatA), or mock treated (−LatA) at 24°C for 30 min. Cells were fixed and stained with Rhodamine-Phalloidin to visualize the actin cytoskeleton. Representative images of cells examined by fluorescence microscopy in the green (GFP) and red (Rh.-Phalloidin) channels are shown. (B) Mmm1p-GFP–expressing strains YAAH1, which contains mtDNA (ρ+) and YAAH2, which lacks mtDNA (ρ°), were stained with 1 μg/ml DAPI to visualize the mtDNA. Although DAPI treatment of living cells preferentially stains mtDNA, low-level staining of nuclear DNA can be seen when images are overexposed or no mtDNA is present. Bars: 2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199622&req=5

Figure 3: Mmm1p maintains a punctate localization in the absence of actin or mtDNA. (A) YAAH1, which expresses Mmm1p-GFP, was grown to early log phase and treated with Latrunculin A (+LatA), or mock treated (−LatA) at 24°C for 30 min. Cells were fixed and stained with Rhodamine-Phalloidin to visualize the actin cytoskeleton. Representative images of cells examined by fluorescence microscopy in the green (GFP) and red (Rh.-Phalloidin) channels are shown. (B) Mmm1p-GFP–expressing strains YAAH1, which contains mtDNA (ρ+) and YAAH2, which lacks mtDNA (ρ°), were stained with 1 μg/ml DAPI to visualize the mtDNA. Although DAPI treatment of living cells preferentially stains mtDNA, low-level staining of nuclear DNA can be seen when images are overexposed or no mtDNA is present. Bars: 2 μm.
Mentions: Yeast mitochondria have been shown to interact with actin filaments (Drubin et al. 1993; Smith et al. 1995) and Mmm1p appears to be important for this interaction (Boldogh et al. 1998). Nevertheless, an intact actin cytoskeleton was not required for Mmm1p distribution. Mmm1p-GFP–containing cells were treated with Latrunculin A to disrupt the actin cytoskeleton (Ayscough et al. 1997) and examined by fluorescence microscopy (Fig. 3 A). Under conditions where normal actin cables and patches cannot be seen by rhodamine-phalloidin staining, the same number of Mmm1p-GFP dots were still present.

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