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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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Mitochondrial membrane vesicles containing Mmm1p are located in a fraction intermediate in density to outer and inner membrane vesicles. Mitochondria were isolated from YSB107 (Burgess et al. 1994), which expresses the Mmm1p-HA fusion protein. Mitochondria were sonicated and membrane vesicles were separated on sucrose gradients (Pon et al. 1989). Gradient fractions were collected and analyzed by Western blotting using antibodies to Om45p, an outer membrane protein, the β-subunit of the F1-ATPase (F1β), an inner membrane protein, and HA (Mmm1p-HA). Lane 1 represents the top of the gradient.
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Figure 8: Mitochondrial membrane vesicles containing Mmm1p are located in a fraction intermediate in density to outer and inner membrane vesicles. Mitochondria were isolated from YSB107 (Burgess et al. 1994), which expresses the Mmm1p-HA fusion protein. Mitochondria were sonicated and membrane vesicles were separated on sucrose gradients (Pon et al. 1989). Gradient fractions were collected and analyzed by Western blotting using antibodies to Om45p, an outer membrane protein, the β-subunit of the F1-ATPase (F1β), an inner membrane protein, and HA (Mmm1p-HA). Lane 1 represents the top of the gradient.

Mentions: Since we find that Mmm1p, an outer membrane protein, is located adjacent to matrix-localized mtDNA nucleoids, our results raise the possibility that Mmm1p resides in contact sites. Contact sites, or regions of close association between the mitochondrial outer and inner membranes, have long been observed in electron micrographs of mitochondria (Hackenbrock 1968; Bereiter-Hahn 1990; Bereiter-Hahn and Voth 1994). Pon et al. 1989 showed that mitochondrial membrane vesicles enriched in contact sites could be isolated on sucrose density gradients. This fraction contained many paired vesicles, with one vesicle containing inner membrane proteins and the other derived from the outer membrane (Pon et al. 1989). Using this procedure, we find that membrane vesicles that contain Mmm1p are located in a fraction intermediate in density to outer and inner membrane vesicles (Fig. 8). Mitochondria from cells expressing an Mmm1p-HA fusion protein were sonicated, and separated on sucrose gradients. Western blots of gradient fractions showed that the outer membrane protein OM45p was located near the top of the gradient (Fig. 8, Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8), and the β subunit of the F1-ATPase, an inner membrane protein, was found in more dense fractions (Fig. 8, Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). Mmm1p-HA–containing vesicles were located between the outer and inner membrane fractions (Fig. 8, Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). Our results thus suggest that Mmm1p is located in contact sites.


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)

Mitochondrial membrane vesicles containing Mmm1p are located in a fraction intermediate in density to outer and inner membrane vesicles. Mitochondria were isolated from YSB107 (Burgess et al. 1994), which expresses the Mmm1p-HA fusion protein. Mitochondria were sonicated and membrane vesicles were separated on sucrose gradients (Pon et al. 1989). Gradient fractions were collected and analyzed by Western blotting using antibodies to Om45p, an outer membrane protein, the β-subunit of the F1-ATPase (F1β), an inner membrane protein, and HA (Mmm1p-HA). Lane 1 represents the top of the gradient.
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Related In: Results  -  Collection

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Figure 8: Mitochondrial membrane vesicles containing Mmm1p are located in a fraction intermediate in density to outer and inner membrane vesicles. Mitochondria were isolated from YSB107 (Burgess et al. 1994), which expresses the Mmm1p-HA fusion protein. Mitochondria were sonicated and membrane vesicles were separated on sucrose gradients (Pon et al. 1989). Gradient fractions were collected and analyzed by Western blotting using antibodies to Om45p, an outer membrane protein, the β-subunit of the F1-ATPase (F1β), an inner membrane protein, and HA (Mmm1p-HA). Lane 1 represents the top of the gradient.
Mentions: Since we find that Mmm1p, an outer membrane protein, is located adjacent to matrix-localized mtDNA nucleoids, our results raise the possibility that Mmm1p resides in contact sites. Contact sites, or regions of close association between the mitochondrial outer and inner membranes, have long been observed in electron micrographs of mitochondria (Hackenbrock 1968; Bereiter-Hahn 1990; Bereiter-Hahn and Voth 1994). Pon et al. 1989 showed that mitochondrial membrane vesicles enriched in contact sites could be isolated on sucrose density gradients. This fraction contained many paired vesicles, with one vesicle containing inner membrane proteins and the other derived from the outer membrane (Pon et al. 1989). Using this procedure, we find that membrane vesicles that contain Mmm1p are located in a fraction intermediate in density to outer and inner membrane vesicles (Fig. 8). Mitochondria from cells expressing an Mmm1p-HA fusion protein were sonicated, and separated on sucrose gradients. Western blots of gradient fractions showed that the outer membrane protein OM45p was located near the top of the gradient (Fig. 8, Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8), and the β subunit of the F1-ATPase, an inner membrane protein, was found in more dense fractions (Fig. 8, Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). Mmm1p-HA–containing vesicles were located between the outer and inner membrane fractions (Fig. 8, Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). Our results thus suggest that Mmm1p is located in contact sites.

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