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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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mmm1-1 cells lose mtDNA at the nonpermissive temperature. (A) Wild-type strain RJ485 and mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times. Total DNA was isolated, Southern blotted, and probed with 32P-labeled mitochondrial COX2 gene. After normalization to the levels of hybridization to the nuclear-encoded TIM23 gene, relative amounts of mtDNA in wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells was plotted. (B) ade2 MMM1 strain RJ485 and ade2 mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times and dilutions of cells were plated onto YEPD plates. After growth at 24°C for 3–5 d, the number of white (lacking functional mtDNA) and red (with functional mtDNA) colonies were determined. The percentage of white colonies using wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells is indicated. (C) Wild-type and mmm1-1 cells were grown at 24°C, and then shifted to 37°C for the indicated times, stained with 1 μg/ml DAPI for 15 min, and examined by fluorescence microscopy. At each time point, 50 cells were examined and the percentage of cells with mtDNA is indicated. Wild-type (□, solid line) and mmm1-1 (♦, dotted line).
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Figure 4: mmm1-1 cells lose mtDNA at the nonpermissive temperature. (A) Wild-type strain RJ485 and mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times. Total DNA was isolated, Southern blotted, and probed with 32P-labeled mitochondrial COX2 gene. After normalization to the levels of hybridization to the nuclear-encoded TIM23 gene, relative amounts of mtDNA in wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells was plotted. (B) ade2 MMM1 strain RJ485 and ade2 mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times and dilutions of cells were plated onto YEPD plates. After growth at 24°C for 3–5 d, the number of white (lacking functional mtDNA) and red (with functional mtDNA) colonies were determined. The percentage of white colonies using wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells is indicated. (C) Wild-type and mmm1-1 cells were grown at 24°C, and then shifted to 37°C for the indicated times, stained with 1 μg/ml DAPI for 15 min, and examined by fluorescence microscopy. At each time point, 50 cells were examined and the percentage of cells with mtDNA is indicated. Wild-type (□, solid line) and mmm1-1 (♦, dotted line).

Mentions: Because Mmm1p-GFP is adjacent to mtDNA nucleoids, we tested the hypothesis that Mmm1p is required to maintain mtDNA, using the temperature-sensitive mmm1-1 mutant (Burgess et al. 1994). mmm1-1 cells have normal mitochondrial shape at 24°C, but lose mitochondrial structure at 37°C. To determine the rate of mtDNA loss in mmm1 mutants, we grew mmm1-1 cells in glucose-containing medium at 24°C and shifted cells to 37°C for the times indicated. At each time point, total DNA was isolated, transferred to filters by Southern blotting (Fig. 4 A) or dot blotting (A. Aiken Hobbs, unpublished observations), and probed for the mtDNA-encoded COX2 gene (Maniatis et al. 1982). We found that mmm1-1 cells rapidly lost mitochondrial DNA at nonpermissive temperature, whereas mtDNA content in wild-type cells remained constant. By 24 h, half of the mmm1-1 cells no longer contained mtDNA and, by 48 h, mtDNA was undetectable by blotting. Normal mitochondrial shape was lost within 60–90 min of the temperature shift (data not shown). mtDNA loss, therefore, appeared to be secondary to the change in mitochondrial morphology.


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)

mmm1-1 cells lose mtDNA at the nonpermissive temperature. (A) Wild-type strain RJ485 and mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times. Total DNA was isolated, Southern blotted, and probed with 32P-labeled mitochondrial COX2 gene. After normalization to the levels of hybridization to the nuclear-encoded TIM23 gene, relative amounts of mtDNA in wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells was plotted. (B) ade2 MMM1 strain RJ485 and ade2 mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times and dilutions of cells were plated onto YEPD plates. After growth at 24°C for 3–5 d, the number of white (lacking functional mtDNA) and red (with functional mtDNA) colonies were determined. The percentage of white colonies using wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells is indicated. (C) Wild-type and mmm1-1 cells were grown at 24°C, and then shifted to 37°C for the indicated times, stained with 1 μg/ml DAPI for 15 min, and examined by fluorescence microscopy. At each time point, 50 cells were examined and the percentage of cells with mtDNA is indicated. Wild-type (□, solid line) and mmm1-1 (♦, dotted line).
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Figure 4: mmm1-1 cells lose mtDNA at the nonpermissive temperature. (A) Wild-type strain RJ485 and mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times. Total DNA was isolated, Southern blotted, and probed with 32P-labeled mitochondrial COX2 gene. After normalization to the levels of hybridization to the nuclear-encoded TIM23 gene, relative amounts of mtDNA in wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells was plotted. (B) ade2 MMM1 strain RJ485 and ade2 mmm1-1 strain YSB105 were grown at 24°C in YEPD medium, and then shifted to 37°C for the indicated times and dilutions of cells were plated onto YEPD plates. After growth at 24°C for 3–5 d, the number of white (lacking functional mtDNA) and red (with functional mtDNA) colonies were determined. The percentage of white colonies using wild-type (□, solid line) and mmm1-1 (♦, dotted line) cells is indicated. (C) Wild-type and mmm1-1 cells were grown at 24°C, and then shifted to 37°C for the indicated times, stained with 1 μg/ml DAPI for 15 min, and examined by fluorescence microscopy. At each time point, 50 cells were examined and the percentage of cells with mtDNA is indicated. Wild-type (□, solid line) and mmm1-1 (♦, dotted line).
Mentions: Because Mmm1p-GFP is adjacent to mtDNA nucleoids, we tested the hypothesis that Mmm1p is required to maintain mtDNA, using the temperature-sensitive mmm1-1 mutant (Burgess et al. 1994). mmm1-1 cells have normal mitochondrial shape at 24°C, but lose mitochondrial structure at 37°C. To determine the rate of mtDNA loss in mmm1 mutants, we grew mmm1-1 cells in glucose-containing medium at 24°C and shifted cells to 37°C for the times indicated. At each time point, total DNA was isolated, transferred to filters by Southern blotting (Fig. 4 A) or dot blotting (A. Aiken Hobbs, unpublished observations), and probed for the mtDNA-encoded COX2 gene (Maniatis et al. 1982). We found that mmm1-1 cells rapidly lost mitochondrial DNA at nonpermissive temperature, whereas mtDNA content in wild-type cells remained constant. By 24 h, half of the mmm1-1 cells no longer contained mtDNA and, by 48 h, mtDNA was undetectable by blotting. Normal mitochondrial shape was lost within 60–90 min of the temperature shift (data not shown). mtDNA loss, therefore, appeared to be secondary to the change in mitochondrial morphology.

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