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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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Mgm1p with a mutated GTP-binding site fails to complement growth or mitochondrial inheritance defects of mgm1- cells.  (A) Detection of Mgm1p expressed from the S224N mutant allele. Yeast cells with an mgm1- mutation (derived from strain  MYY973) harboring plasmids encoding either wild-type MGM1 (left) or mgm1-S224N (right) were grown in minimal medium without  histidine. Cells were homogenized by glass-bead lysis, and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies  specific for Mgm1p. (B) Cells with an mgm1- mutation (derived from strain MYY973) harboring plasmids encoding wild-type  MGM1 (pRS313-MGM1), mgm1-S224N (p313-MGM1-S224N), or vector alone (pRS313) were cultured in different sectors of a YPG-agar plate at 30°C for 48 h. (C) mdm17 cells (strain MYY972) were transformed with plasmids encoding wild-type MGM1 (pRS313-MGM1) or the S224N mutant (pMGM1-S224N) and cultured in minimal medium without histidine at 23°C. Cells were incubated at  37°C for 1 h, stained with DASPMI, and examined by phase-contrast (left of each pair) and fluorescence microscopy (right of each  pair). Two representative cells of each type are shown. Bar, 2 μm.
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Figure 7: Mgm1p with a mutated GTP-binding site fails to complement growth or mitochondrial inheritance defects of mgm1- cells. (A) Detection of Mgm1p expressed from the S224N mutant allele. Yeast cells with an mgm1- mutation (derived from strain MYY973) harboring plasmids encoding either wild-type MGM1 (left) or mgm1-S224N (right) were grown in minimal medium without histidine. Cells were homogenized by glass-bead lysis, and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies specific for Mgm1p. (B) Cells with an mgm1- mutation (derived from strain MYY973) harboring plasmids encoding wild-type MGM1 (pRS313-MGM1), mgm1-S224N (p313-MGM1-S224N), or vector alone (pRS313) were cultured in different sectors of a YPG-agar plate at 30°C for 48 h. (C) mdm17 cells (strain MYY972) were transformed with plasmids encoding wild-type MGM1 (pRS313-MGM1) or the S224N mutant (pMGM1-S224N) and cultured in minimal medium without histidine at 23°C. Cells were incubated at 37°C for 1 h, stained with DASPMI, and examined by phase-contrast (left of each pair) and fluorescence microscopy (right of each pair). Two representative cells of each type are shown. Bar, 2 μm.

Mentions: To explore further the function of Mgm1p, the identity of the mdm17 mutation was determined. The mutant gene contained a single change: a transition of G to A at nucleotide 880, resulting in a change of Glu294 to Lys. This lesion mapped near the conserved, tripartite GTP-binding motif, suggesting a role for this site in Mgm1p function. To test the importance of the GTP-binding site, a mutant form of MGM1 incorporating a change in a conserved residue, S224N, was created (the numbering of this residue is based on the designation of the second methionine in MGM1 as the NH2-terminal residue as described above). The equivalent mutation in the H-ras-GTPase (S17N) eliminates the protein's binding of GTP (Feig and Cooper, 1988), and a corresponding mutation in rat dynamin (S45N) blocks the protein's function in endocytosis (Herskovits et al., 1993). Immunoblot analysis of cellular extracts detected Mgm1p in mgm1- cells harboring the mutant gene (Fig. 7 A). However, the mgm1-S224N gene failed to complement any of the mutant phenotypes of a cell containing an mgm1- lesion (Fig. 7 B and data not shown). In addition, the mgm1-S224N gene did not complement the growth or mitochondrial distribution and morphology defects of the mdm17 mutant cells (Fig. 7 C). These results indicate that an intact GTP-binding domain is essential for Mgm1p function.


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

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

Mgm1p with a mutated GTP-binding site fails to complement growth or mitochondrial inheritance defects of mgm1- cells.  (A) Detection of Mgm1p expressed from the S224N mutant allele. Yeast cells with an mgm1- mutation (derived from strain  MYY973) harboring plasmids encoding either wild-type MGM1 (left) or mgm1-S224N (right) were grown in minimal medium without  histidine. Cells were homogenized by glass-bead lysis, and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies  specific for Mgm1p. (B) Cells with an mgm1- mutation (derived from strain MYY973) harboring plasmids encoding wild-type  MGM1 (pRS313-MGM1), mgm1-S224N (p313-MGM1-S224N), or vector alone (pRS313) were cultured in different sectors of a YPG-agar plate at 30°C for 48 h. (C) mdm17 cells (strain MYY972) were transformed with plasmids encoding wild-type MGM1 (pRS313-MGM1) or the S224N mutant (pMGM1-S224N) and cultured in minimal medium without histidine at 23°C. Cells were incubated at  37°C for 1 h, stained with DASPMI, and examined by phase-contrast (left of each pair) and fluorescence microscopy (right of each  pair). Two representative cells of each type are shown. Bar, 2 μm.
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Figure 7: Mgm1p with a mutated GTP-binding site fails to complement growth or mitochondrial inheritance defects of mgm1- cells. (A) Detection of Mgm1p expressed from the S224N mutant allele. Yeast cells with an mgm1- mutation (derived from strain MYY973) harboring plasmids encoding either wild-type MGM1 (left) or mgm1-S224N (right) were grown in minimal medium without histidine. Cells were homogenized by glass-bead lysis, and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies specific for Mgm1p. (B) Cells with an mgm1- mutation (derived from strain MYY973) harboring plasmids encoding wild-type MGM1 (pRS313-MGM1), mgm1-S224N (p313-MGM1-S224N), or vector alone (pRS313) were cultured in different sectors of a YPG-agar plate at 30°C for 48 h. (C) mdm17 cells (strain MYY972) were transformed with plasmids encoding wild-type MGM1 (pRS313-MGM1) or the S224N mutant (pMGM1-S224N) and cultured in minimal medium without histidine at 23°C. Cells were incubated at 37°C for 1 h, stained with DASPMI, and examined by phase-contrast (left of each pair) and fluorescence microscopy (right of each pair). Two representative cells of each type are shown. Bar, 2 μm.
Mentions: To explore further the function of Mgm1p, the identity of the mdm17 mutation was determined. The mutant gene contained a single change: a transition of G to A at nucleotide 880, resulting in a change of Glu294 to Lys. This lesion mapped near the conserved, tripartite GTP-binding motif, suggesting a role for this site in Mgm1p function. To test the importance of the GTP-binding site, a mutant form of MGM1 incorporating a change in a conserved residue, S224N, was created (the numbering of this residue is based on the designation of the second methionine in MGM1 as the NH2-terminal residue as described above). The equivalent mutation in the H-ras-GTPase (S17N) eliminates the protein's binding of GTP (Feig and Cooper, 1988), and a corresponding mutation in rat dynamin (S45N) blocks the protein's function in endocytosis (Herskovits et al., 1993). Immunoblot analysis of cellular extracts detected Mgm1p in mgm1- cells harboring the mutant gene (Fig. 7 A). However, the mgm1-S224N gene failed to complement any of the mutant phenotypes of a cell containing an mgm1- lesion (Fig. 7 B and data not shown). In addition, the mgm1-S224N gene did not complement the growth or mitochondrial distribution and morphology defects of the mdm17 mutant cells (Fig. 7 C). These results indicate that an intact GTP-binding domain is essential for Mgm1p function.

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