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The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion.

Wong ED, Wagner JA, Scott SV, Okreglak V, Holewinske TJ, Cassidy-Stone A, Nunnari J - J. Cell Biol. (2003)

Bottom Line: In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p.Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane.To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular and Cellular Biology, University of California, Davis, Davis, California, 95616, USA.

ABSTRACT
A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion.

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Determination of the steady-state levels of mutant Mgm1 proteins. JNY845 was transformed with pRS425 vectors containing mgm1 mutants, and strains were grown overnight to log phase. Whole cell extracts were made and analyzed by SDS-PAGE followed by Western blotting as described in the Materials and methods. Western analysis of the cytosolic protein 3-phosphoglycerokinase (PGK) was used as a loading control.
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fig3: Determination of the steady-state levels of mutant Mgm1 proteins. JNY845 was transformed with pRS425 vectors containing mgm1 mutants, and strains were grown overnight to log phase. Whole cell extracts were made and analyzed by SDS-PAGE followed by Western blotting as described in the Materials and methods. Western analysis of the cytosolic protein 3-phosphoglycerokinase (PGK) was used as a loading control.

Mentions: We created mutants predicted to be defective in the GTPase cycle, based on the analysis of dynamin and other GTPase superfamily members, by mutating specific conserved residues in the G1 and G2 nucleotide binding regions of Mgm1p. Conserved residues in the G1 region, K223 and S224, and in the G2 region, T244, were mutated to alanine in Mgm1p. We first examined the expression of these mutant forms of Mgm1p in Δmgm1 cells by Western blotting. Interestingly, only Mgm1S224A and Mgm1T244A were detected in cells, suggesting that the nucleotide binding state of Mgm1p affects its stability in vivo (Fig. 3; unpublished data).


The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion.

Wong ED, Wagner JA, Scott SV, Okreglak V, Holewinske TJ, Cassidy-Stone A, Nunnari J - J. Cell Biol. (2003)

Determination of the steady-state levels of mutant Mgm1 proteins. JNY845 was transformed with pRS425 vectors containing mgm1 mutants, and strains were grown overnight to log phase. Whole cell extracts were made and analyzed by SDS-PAGE followed by Western blotting as described in the Materials and methods. Western analysis of the cytosolic protein 3-phosphoglycerokinase (PGK) was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172654&req=5

fig3: Determination of the steady-state levels of mutant Mgm1 proteins. JNY845 was transformed with pRS425 vectors containing mgm1 mutants, and strains were grown overnight to log phase. Whole cell extracts were made and analyzed by SDS-PAGE followed by Western blotting as described in the Materials and methods. Western analysis of the cytosolic protein 3-phosphoglycerokinase (PGK) was used as a loading control.
Mentions: We created mutants predicted to be defective in the GTPase cycle, based on the analysis of dynamin and other GTPase superfamily members, by mutating specific conserved residues in the G1 and G2 nucleotide binding regions of Mgm1p. Conserved residues in the G1 region, K223 and S224, and in the G2 region, T244, were mutated to alanine in Mgm1p. We first examined the expression of these mutant forms of Mgm1p in Δmgm1 cells by Western blotting. Interestingly, only Mgm1S224A and Mgm1T244A were detected in cells, suggesting that the nucleotide binding state of Mgm1p affects its stability in vivo (Fig. 3; unpublished data).

Bottom Line: In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p.Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane.To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular and Cellular Biology, University of California, Davis, Davis, California, 95616, USA.

ABSTRACT
A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion.

Show MeSH
Related in: MedlinePlus