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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 adjacent to mitochondrial DNA nucleoids. Mmm1p-GFP–containing strain YAAH1 was grown to early log phase, stained with DAPI, and examined using a DeltaVision microscope. DAPI staining of live cells preferentially stains mtDNA, and the nucleus is only poorly stained (Williamson and Fennell 1979). Shown are all 15 deconvolved z sections, which have been flattened to a single image. (A) DIC; (B) Mmm1p-GFP fluorescence; (C) DAPI fluorescence; (D) merged images of B and C. (E–H) The images in D were processed using the DeltaVision model building tool, which removes background and shows fluorescent signals as solid objects for easier viewing. Four views of the 3-D model, each rotated 0° (E), 65° (F), 130° (G), and 190° (H) with respect to each other. Bar: 2 μm.
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Figure 2: Mmm1p is adjacent to mitochondrial DNA nucleoids. Mmm1p-GFP–containing strain YAAH1 was grown to early log phase, stained with DAPI, and examined using a DeltaVision microscope. DAPI staining of live cells preferentially stains mtDNA, and the nucleus is only poorly stained (Williamson and Fennell 1979). Shown are all 15 deconvolved z sections, which have been flattened to a single image. (A) DIC; (B) Mmm1p-GFP fluorescence; (C) DAPI fluorescence; (D) merged images of B and C. (E–H) The images in D were processed using the DeltaVision model building tool, which removes background and shows fluorescent signals as solid objects for easier viewing. Four views of the 3-D model, each rotated 0° (E), 65° (F), 130° (G), and 190° (H) with respect to each other. Bar: 2 μm.

Mentions: In yeast cells, mtDNA is organized into ∼10–20 separate DNA-protein complexes called nucleoids (Fig. 2 C; Miyakawa et al. 1987; Kaufman et al. 2000). Since the punctate distribution of mtDNA is similar to the pattern we found for Mmm1p, we stained Mmm1p-GFP–containing cells with DAPI and examined cells by fluorescence microscopy. Strikingly, the majority of Mmm1p-GFP was associated with mtDNA nucleoids (Fig. 2, A–D; also see online movie). When a total of 32 cells were examined using the DeltaVision microscope, deconvolved images and 3-D reconstruction showed that >87% of Mmm1p-GFP structures (186 of 213 GFP-containing dots) were clearly associated with mtDNA. The remaining 27 Mmm1p-GFP structures may also have been adjacent to mtDNA, but our images did not reveal this unambiguously. It is important to note that while Mmm1p-GFP and mtDNA are adjacent, they are clearly in separate structures. In most of the merged images (Fig. 2 D), the Mmm1p-GFP and nucleoids were seen as twin, dot-like structures, one red and one green. Occasionally, a single yellow dot was seen, which probably represented a pair of dots viewed from the top or bottom, instead of from the side. Supporting this idea, 3-D reconstruction of images processed with the DeltaVision model building tool, which removes background and shows fluorescent signals as solid objects for easier viewing, also confirmed the side-by-side location of Mmm1p and mtDNA (Fig. 2, E–H).


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 adjacent to mitochondrial DNA nucleoids. Mmm1p-GFP–containing strain YAAH1 was grown to early log phase, stained with DAPI, and examined using a DeltaVision microscope. DAPI staining of live cells preferentially stains mtDNA, and the nucleus is only poorly stained (Williamson and Fennell 1979). Shown are all 15 deconvolved z sections, which have been flattened to a single image. (A) DIC; (B) Mmm1p-GFP fluorescence; (C) DAPI fluorescence; (D) merged images of B and C. (E–H) The images in D were processed using the DeltaVision model building tool, which removes background and shows fluorescent signals as solid objects for easier viewing. Four views of the 3-D model, each rotated 0° (E), 65° (F), 130° (G), and 190° (H) with respect to each other. Bar: 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199622&req=5

Figure 2: Mmm1p is adjacent to mitochondrial DNA nucleoids. Mmm1p-GFP–containing strain YAAH1 was grown to early log phase, stained with DAPI, and examined using a DeltaVision microscope. DAPI staining of live cells preferentially stains mtDNA, and the nucleus is only poorly stained (Williamson and Fennell 1979). Shown are all 15 deconvolved z sections, which have been flattened to a single image. (A) DIC; (B) Mmm1p-GFP fluorescence; (C) DAPI fluorescence; (D) merged images of B and C. (E–H) The images in D were processed using the DeltaVision model building tool, which removes background and shows fluorescent signals as solid objects for easier viewing. Four views of the 3-D model, each rotated 0° (E), 65° (F), 130° (G), and 190° (H) with respect to each other. Bar: 2 μm.
Mentions: In yeast cells, mtDNA is organized into ∼10–20 separate DNA-protein complexes called nucleoids (Fig. 2 C; Miyakawa et al. 1987; Kaufman et al. 2000). Since the punctate distribution of mtDNA is similar to the pattern we found for Mmm1p, we stained Mmm1p-GFP–containing cells with DAPI and examined cells by fluorescence microscopy. Strikingly, the majority of Mmm1p-GFP was associated with mtDNA nucleoids (Fig. 2, A–D; also see online movie). When a total of 32 cells were examined using the DeltaVision microscope, deconvolved images and 3-D reconstruction showed that >87% of Mmm1p-GFP structures (186 of 213 GFP-containing dots) were clearly associated with mtDNA. The remaining 27 Mmm1p-GFP structures may also have been adjacent to mtDNA, but our images did not reveal this unambiguously. It is important to note that while Mmm1p-GFP and mtDNA are adjacent, they are clearly in separate structures. In most of the merged images (Fig. 2 D), the Mmm1p-GFP and nucleoids were seen as twin, dot-like structures, one red and one green. Occasionally, a single yellow dot was seen, which probably represented a pair of dots viewed from the top or bottom, instead of from the side. Supporting this idea, 3-D reconstruction of images processed with the DeltaVision model building tool, which removes background and shows fluorescent signals as solid objects for easier viewing, also confirmed the side-by-side location of Mmm1p and mtDNA (Fig. 2, E–H).

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