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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 is localized to the mitochondrial outer membrane. (A) Wild-type cells (strain MYY290) (MGM1) and  mgm1- cells (strain MYY974) (mgm1Δ) were grown on YPD  at 30°C. Wild-type cells (strain MYY290) harboring a multicopy  plasmid encoding MGM1 (p423-MGM1) (2 μ, MGM1) were  grown at 30°C on minimal glucose medium without histidine. Extracts of total cellular proteins were prepared by glass bead lysis  and subjected to SDS-PAGE and immunoblot analysis using antibodies specific for the COOH terminus of Mgm1p. The mobilities of molecular mass markers are indicated in kilodaltons at the  left. (B) Subcellular fractions were prepared by differential centrifugation of homogenate from wild-type cells (MYY291) grown  on semisynthetic lactate medium at 30°C. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. Fractions  were probed with anti-Mgm1p antibodies (top) or with antibodies specific for the mitochondrial outer membrane protein, OM45  (bottom). Subcellular fractions are all of the following: T, total  cell homogenate; L, low speed pellet; M, mitochondrial fraction;  I, intermediate speed pellet; H, high speed pellet; and C, cytosolic  fraction. (C) Mgm1p is enriched in the mitochondrial outer membrane. Mitochondria from wild-type cells (strain MYY290) were  fractionated by osmotic shock followed by sucrose density gradient centrifugation to separate outer and inner membranes.  Equivalent amounts (20 μg) of protein from outer (OM) and inner (IM) membrane fractions were analyzed by SDS-PAGE and  immunoblot analysis. Fractions were analyzed with anti-Mgm1p  antibodies (top), anti-F1β antiserum (middle), and anti-OM45  (bottom). (D) Mitochondria isolated from wild-type cells  (MYY290) grown on semisynthetic lactate medium at 30°C were  treated with varying concentrations of trypsin for 10 min at 4°C in  the presence (+) or absence (−) of 1% Triton X-100. Samples  were analyzed by SDS-PAGE and immunoblotting with antibodies against Mgm1p (top), Tom70p, a protein of the mitochondrial  outer membrane (middle), and Mas2p, a matrix protein (bottom).
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Figure 4: Mgm1p is localized to the mitochondrial outer membrane. (A) Wild-type cells (strain MYY290) (MGM1) and mgm1- cells (strain MYY974) (mgm1Δ) were grown on YPD at 30°C. Wild-type cells (strain MYY290) harboring a multicopy plasmid encoding MGM1 (p423-MGM1) (2 μ, MGM1) were grown at 30°C on minimal glucose medium without histidine. Extracts of total cellular proteins were prepared by glass bead lysis and subjected to SDS-PAGE and immunoblot analysis using antibodies specific for the COOH terminus of Mgm1p. The mobilities of molecular mass markers are indicated in kilodaltons at the left. (B) Subcellular fractions were prepared by differential centrifugation of homogenate from wild-type cells (MYY291) grown on semisynthetic lactate medium at 30°C. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. Fractions were probed with anti-Mgm1p antibodies (top) or with antibodies specific for the mitochondrial outer membrane protein, OM45 (bottom). Subcellular fractions are all of the following: T, total cell homogenate; L, low speed pellet; M, mitochondrial fraction; I, intermediate speed pellet; H, high speed pellet; and C, cytosolic fraction. (C) Mgm1p is enriched in the mitochondrial outer membrane. Mitochondria from wild-type cells (strain MYY290) were fractionated by osmotic shock followed by sucrose density gradient centrifugation to separate outer and inner membranes. Equivalent amounts (20 μg) of protein from outer (OM) and inner (IM) membrane fractions were analyzed by SDS-PAGE and immunoblot analysis. Fractions were analyzed with anti-Mgm1p antibodies (top), anti-F1β antiserum (middle), and anti-OM45 (bottom). (D) Mitochondria isolated from wild-type cells (MYY290) grown on semisynthetic lactate medium at 30°C were treated with varying concentrations of trypsin for 10 min at 4°C in the presence (+) or absence (−) of 1% Triton X-100. Samples were analyzed by SDS-PAGE and immunoblotting with antibodies against Mgm1p (top), Tom70p, a protein of the mitochondrial outer membrane (middle), and Mas2p, a matrix protein (bottom).

Mentions: Previously reported phenotypes of the mgm1 mutant, together with the resemblance of the protein's predicted NH2 terminus to a mitochondrial targeting sequence, suggested that Mgm1p is a mitochondrial protein (Guan et al., 1993). However, direct evidence of the protein's subcellular location was lacking. To determine the functional location of Mgm1p, antibodies were generated that were specific for a peptide corresponding to the extreme COOH terminus. These antibodies were used to detect the protein in subcellular fractions. Immunoblotting of total cellular extracts indicated that the affinity-purified antibodies recognized two polypeptides of ∼100 and 90 kD (Fig. 4 A). Neither of these species was apparent in mgm1- cells (Fig. 4 A), indicating that both polypeptides are products of the MGM1 gene. Furthermore, both species displayed substantially increased levels in cells harboring a multicopy (2 μ) plasmid encoding MGM1 (Fig. 4 A). These increased levels had no apparent effect on mitochondrial distribution and morphology (data not shown).


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 is localized to the mitochondrial outer membrane. (A) Wild-type cells (strain MYY290) (MGM1) and  mgm1- cells (strain MYY974) (mgm1Δ) were grown on YPD  at 30°C. Wild-type cells (strain MYY290) harboring a multicopy  plasmid encoding MGM1 (p423-MGM1) (2 μ, MGM1) were  grown at 30°C on minimal glucose medium without histidine. Extracts of total cellular proteins were prepared by glass bead lysis  and subjected to SDS-PAGE and immunoblot analysis using antibodies specific for the COOH terminus of Mgm1p. The mobilities of molecular mass markers are indicated in kilodaltons at the  left. (B) Subcellular fractions were prepared by differential centrifugation of homogenate from wild-type cells (MYY291) grown  on semisynthetic lactate medium at 30°C. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. Fractions  were probed with anti-Mgm1p antibodies (top) or with antibodies specific for the mitochondrial outer membrane protein, OM45  (bottom). Subcellular fractions are all of the following: T, total  cell homogenate; L, low speed pellet; M, mitochondrial fraction;  I, intermediate speed pellet; H, high speed pellet; and C, cytosolic  fraction. (C) Mgm1p is enriched in the mitochondrial outer membrane. Mitochondria from wild-type cells (strain MYY290) were  fractionated by osmotic shock followed by sucrose density gradient centrifugation to separate outer and inner membranes.  Equivalent amounts (20 μg) of protein from outer (OM) and inner (IM) membrane fractions were analyzed by SDS-PAGE and  immunoblot analysis. Fractions were analyzed with anti-Mgm1p  antibodies (top), anti-F1β antiserum (middle), and anti-OM45  (bottom). (D) Mitochondria isolated from wild-type cells  (MYY290) grown on semisynthetic lactate medium at 30°C were  treated with varying concentrations of trypsin for 10 min at 4°C in  the presence (+) or absence (−) of 1% Triton X-100. Samples  were analyzed by SDS-PAGE and immunoblotting with antibodies against Mgm1p (top), Tom70p, a protein of the mitochondrial  outer membrane (middle), and Mas2p, a matrix protein (bottom).
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Figure 4: Mgm1p is localized to the mitochondrial outer membrane. (A) Wild-type cells (strain MYY290) (MGM1) and mgm1- cells (strain MYY974) (mgm1Δ) were grown on YPD at 30°C. Wild-type cells (strain MYY290) harboring a multicopy plasmid encoding MGM1 (p423-MGM1) (2 μ, MGM1) were grown at 30°C on minimal glucose medium without histidine. Extracts of total cellular proteins were prepared by glass bead lysis and subjected to SDS-PAGE and immunoblot analysis using antibodies specific for the COOH terminus of Mgm1p. The mobilities of molecular mass markers are indicated in kilodaltons at the left. (B) Subcellular fractions were prepared by differential centrifugation of homogenate from wild-type cells (MYY291) grown on semisynthetic lactate medium at 30°C. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. Fractions were probed with anti-Mgm1p antibodies (top) or with antibodies specific for the mitochondrial outer membrane protein, OM45 (bottom). Subcellular fractions are all of the following: T, total cell homogenate; L, low speed pellet; M, mitochondrial fraction; I, intermediate speed pellet; H, high speed pellet; and C, cytosolic fraction. (C) Mgm1p is enriched in the mitochondrial outer membrane. Mitochondria from wild-type cells (strain MYY290) were fractionated by osmotic shock followed by sucrose density gradient centrifugation to separate outer and inner membranes. Equivalent amounts (20 μg) of protein from outer (OM) and inner (IM) membrane fractions were analyzed by SDS-PAGE and immunoblot analysis. Fractions were analyzed with anti-Mgm1p antibodies (top), anti-F1β antiserum (middle), and anti-OM45 (bottom). (D) Mitochondria isolated from wild-type cells (MYY290) grown on semisynthetic lactate medium at 30°C were treated with varying concentrations of trypsin for 10 min at 4°C in the presence (+) or absence (−) of 1% Triton X-100. Samples were analyzed by SDS-PAGE and immunoblotting with antibodies against Mgm1p (top), Tom70p, a protein of the mitochondrial outer membrane (middle), and Mas2p, a matrix protein (bottom).
Mentions: Previously reported phenotypes of the mgm1 mutant, together with the resemblance of the protein's predicted NH2 terminus to a mitochondrial targeting sequence, suggested that Mgm1p is a mitochondrial protein (Guan et al., 1993). However, direct evidence of the protein's subcellular location was lacking. To determine the functional location of Mgm1p, antibodies were generated that were specific for a peptide corresponding to the extreme COOH terminus. These antibodies were used to detect the protein in subcellular fractions. Immunoblotting of total cellular extracts indicated that the affinity-purified antibodies recognized two polypeptides of ∼100 and 90 kD (Fig. 4 A). Neither of these species was apparent in mgm1- cells (Fig. 4 A), indicating that both polypeptides are products of the MGM1 gene. Furthermore, both species displayed substantially increased levels in cells harboring a multicopy (2 μ) plasmid encoding MGM1 (Fig. 4 A). These increased levels had no apparent effect on mitochondrial distribution and morphology (data not shown).

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