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The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance.

Shepard KA, Yaffe MP - J. Cell Biol. (1999)

Bottom Line: It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene.Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex.These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California, San Diego, La Jolla, California 92093-0347, USA.

ABSTRACT
The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1- mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

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Dominant-negative effects of Mgm1p mutated in the GTP-binding site. Haploid cells with an mgm1- mutation harboring  multicopy plasmids encoding either wild-type (p423-MGM1) or S224N mutant (p423-MGM1-S224N) forms of MGM1 were crossed to  wild-type haploid cells. Mitochondria in zygotes were visualized by staining with DASPMI and fluorescence microscopy. (A) Phase-contrast and fluorescence images of two representative budded zygotes harboring wild-type (upper panels) or mutant (lower panels)  versions of MGM1. Bar, 2 μm. (B) Quantitation of zygote phenotypes. Stained zygotes (budded and unbudded) were scored for mitochondrial aggregation, and budded zygotes were scored for the presence of stained mitochondria in the buds. At least 300 zygotes were  counted for each sample.
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Figure 8: Dominant-negative effects of Mgm1p mutated in the GTP-binding site. Haploid cells with an mgm1- mutation harboring multicopy plasmids encoding either wild-type (p423-MGM1) or S224N mutant (p423-MGM1-S224N) forms of MGM1 were crossed to wild-type haploid cells. Mitochondria in zygotes were visualized by staining with DASPMI and fluorescence microscopy. (A) Phase-contrast and fluorescence images of two representative budded zygotes harboring wild-type (upper panels) or mutant (lower panels) versions of MGM1. Bar, 2 μm. (B) Quantitation of zygote phenotypes. Stained zygotes (budded and unbudded) were scored for mitochondrial aggregation, and budded zygotes were scored for the presence of stained mitochondria in the buds. At least 300 zygotes were counted for each sample.

Mentions: Overexpression of dynamin family members mutated in critical residues of the GTP-binding site was previously found to cause dominant-negative effects in wild-type cells (Warnock and Schmid, 1996). To test whether this is also true for MGM1, high-copy plasmids encoding either wild-type MGM1 or mgm1-S224N were transformed into wild-type cells. Only a small number of transformants harboring the mutant gene was recovered, and Western blot analysis indicated that the mutant protein was not overexpressed in these cells (perhaps reflecting a regulatory or suppression phenomenon). However, both wild-type and mutant proteins were overexpressed in mgm1- cells harboring the respective plasmids (data not shown). These transformed cells were mated to wild-type cells, and the effect of overexpression in the resulting zygotes was analyzed by fluorescence microscopy. High levels of wild-type MGM1 had no apparent effect on mitochondrial distribution or morphology (Fig. 8 A), but zygotes with elevated levels of the mutant protein displayed defects very similar to those caused by the original mdm17 mutation (Fig. 8 A). In particular, 90% of the latter zygotes displayed aggregated mitochondria (Fig. 8 B), and 52% of the buds produced by these zygotes contained no mitochondria (Fig. 8 B). These results demonstrate that the mgm1-S224N mutant gene product confers dominant-negative effects on mitochondrial distribution and morphology.


The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance.

Shepard KA, Yaffe MP - J. Cell Biol. (1999)

Dominant-negative effects of Mgm1p mutated in the GTP-binding site. Haploid cells with an mgm1- mutation harboring  multicopy plasmids encoding either wild-type (p423-MGM1) or S224N mutant (p423-MGM1-S224N) forms of MGM1 were crossed to  wild-type haploid cells. Mitochondria in zygotes were visualized by staining with DASPMI and fluorescence microscopy. (A) Phase-contrast and fluorescence images of two representative budded zygotes harboring wild-type (upper panels) or mutant (lower panels)  versions of MGM1. Bar, 2 μm. (B) Quantitation of zygote phenotypes. Stained zygotes (budded and unbudded) were scored for mitochondrial aggregation, and budded zygotes were scored for the presence of stained mitochondria in the buds. At least 300 zygotes were  counted for each sample.
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Related In: Results  -  Collection

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Figure 8: Dominant-negative effects of Mgm1p mutated in the GTP-binding site. Haploid cells with an mgm1- mutation harboring multicopy plasmids encoding either wild-type (p423-MGM1) or S224N mutant (p423-MGM1-S224N) forms of MGM1 were crossed to wild-type haploid cells. Mitochondria in zygotes were visualized by staining with DASPMI and fluorescence microscopy. (A) Phase-contrast and fluorescence images of two representative budded zygotes harboring wild-type (upper panels) or mutant (lower panels) versions of MGM1. Bar, 2 μm. (B) Quantitation of zygote phenotypes. Stained zygotes (budded and unbudded) were scored for mitochondrial aggregation, and budded zygotes were scored for the presence of stained mitochondria in the buds. At least 300 zygotes were counted for each sample.
Mentions: Overexpression of dynamin family members mutated in critical residues of the GTP-binding site was previously found to cause dominant-negative effects in wild-type cells (Warnock and Schmid, 1996). To test whether this is also true for MGM1, high-copy plasmids encoding either wild-type MGM1 or mgm1-S224N were transformed into wild-type cells. Only a small number of transformants harboring the mutant gene was recovered, and Western blot analysis indicated that the mutant protein was not overexpressed in these cells (perhaps reflecting a regulatory or suppression phenomenon). However, both wild-type and mutant proteins were overexpressed in mgm1- cells harboring the respective plasmids (data not shown). These transformed cells were mated to wild-type cells, and the effect of overexpression in the resulting zygotes was analyzed by fluorescence microscopy. High levels of wild-type MGM1 had no apparent effect on mitochondrial distribution or morphology (Fig. 8 A), but zygotes with elevated levels of the mutant protein displayed defects very similar to those caused by the original mdm17 mutation (Fig. 8 A). In particular, 90% of the latter zygotes displayed aggregated mitochondria (Fig. 8 B), and 52% of the buds produced by these zygotes contained no mitochondria (Fig. 8 B). These results demonstrate that the mgm1-S224N mutant gene product confers dominant-negative effects on mitochondrial distribution and morphology.

Bottom Line: It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene.Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex.These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California, San Diego, La Jolla, California 92093-0347, USA.

ABSTRACT
The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1- mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

Show MeSH
Related in: MedlinePlus