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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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Mgm1p is in the IMS compartment in vivo. Mitochondria were isolated from strains expressing Mgm1:tev:3XHAp alone (lane 1), Mgm1:tev:3XHAp and ATP9-TEVp (lane 2), and Mgm1:tev:3XHAp and CYB2-TEVp (lane 3) and analyzed by SDS-PAGE followed by Western blotting with anti-HA and anti-porin antibodies, as a loading control. Asterisk indicates a TEV-specific Mgm1 cleavage product.
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fig5: Mgm1p is in the IMS compartment in vivo. Mitochondria were isolated from strains expressing Mgm1:tev:3XHAp alone (lane 1), Mgm1:tev:3XHAp and ATP9-TEVp (lane 2), and Mgm1:tev:3XHAp and CYB2-TEVp (lane 3) and analyzed by SDS-PAGE followed by Western blotting with anti-HA and anti-porin antibodies, as a loading control. Asterisk indicates a TEV-specific Mgm1 cleavage product.

Mentions: Western blot analysis of mitochondrial fractions isolated from wild-type cells expressing Mgm1:tev:3XHAp alone with anti-HA antibodies revealed two predominant forms of Mgm1:tev:3XHAp, as seen previously in cells expressing both Mgm1:3XHAp and native, untagged Mgm1p (Fig. 5, lane 1). When coexpressed with matrix-TEV, the two Mgm1:tev:3XHAp products remained unchanged (Fig. 5, lane 2). In contrast, an additional, faster-migrating species, estimated at ∼75 kD, was detected with anti-HA antibodies in cells coexpressing Mgm1:tev:3XHAp and IMS-TEV (Fig. 1, lane 3, asterisk). The apparent molecular mass of this species is in agreement with the size of a predicted TEV-dependent cleavage product of Mgm1:tev:3XHAp and indicates that Mgm1p was accessible only to the IMS-targeted version of TEV. This observation confirms our previously published results that Mgm1p is localized to the intermembrane space and is in agreement with the recently published localization of the human Mgm1p orthologue, OPA1 (Wong et al., 2000; Olichon et al., 2002).


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)

Mgm1p is in the IMS compartment in vivo. Mitochondria were isolated from strains expressing Mgm1:tev:3XHAp alone (lane 1), Mgm1:tev:3XHAp and ATP9-TEVp (lane 2), and Mgm1:tev:3XHAp and CYB2-TEVp (lane 3) and analyzed by SDS-PAGE followed by Western blotting with anti-HA and anti-porin antibodies, as a loading control. Asterisk indicates a TEV-specific Mgm1 cleavage product.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Mgm1p is in the IMS compartment in vivo. Mitochondria were isolated from strains expressing Mgm1:tev:3XHAp alone (lane 1), Mgm1:tev:3XHAp and ATP9-TEVp (lane 2), and Mgm1:tev:3XHAp and CYB2-TEVp (lane 3) and analyzed by SDS-PAGE followed by Western blotting with anti-HA and anti-porin antibodies, as a loading control. Asterisk indicates a TEV-specific Mgm1 cleavage product.
Mentions: Western blot analysis of mitochondrial fractions isolated from wild-type cells expressing Mgm1:tev:3XHAp alone with anti-HA antibodies revealed two predominant forms of Mgm1:tev:3XHAp, as seen previously in cells expressing both Mgm1:3XHAp and native, untagged Mgm1p (Fig. 5, lane 1). When coexpressed with matrix-TEV, the two Mgm1:tev:3XHAp products remained unchanged (Fig. 5, lane 2). In contrast, an additional, faster-migrating species, estimated at ∼75 kD, was detected with anti-HA antibodies in cells coexpressing Mgm1:tev:3XHAp and IMS-TEV (Fig. 1, lane 3, asterisk). The apparent molecular mass of this species is in agreement with the size of a predicted TEV-dependent cleavage product of Mgm1:tev:3XHAp and indicates that Mgm1p was accessible only to the IMS-targeted version of TEV. This observation confirms our previously published results that Mgm1p is localized to the intermembrane space and is in agreement with the recently published localization of the human Mgm1p orthologue, OPA1 (Wong et al., 2000; Olichon et al., 2002).

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