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UGO1 encodes an outer membrane protein required for mitochondrial fusion.

Sesaki H, Jensen RE - J. Cell Biol. (2001)

Bottom Line: In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents.We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane.Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. hsesaki@jhmi.edu

ABSTRACT
Membrane fusion plays an important role in controlling the shape, number, and distribution of mitochondria. In the yeast Saccharomyces cerevisiae, the outer membrane protein Fzo1p has been shown to mediate mitochondrial fusion. Using a novel genetic screen, we have isolated new mutants defective in the fusion of their mitochondria. One of these mutants, ugo1, shows several similarities to fzo1 mutants. ugo1 cells contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. ugo1 mutants lose mitochondrial DNA (mtDNA). In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents. Fragmentation of mitochondria and loss of mtDNA in ugo1 mutants are rescued by disrupting DNM1, a gene required for mitochondrial division. We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane. Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space. Our results suggest that Ugo1p is a new outer membrane component of the mitochondrial fusion machinery.

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ugo1Δ dnm1Δ cells contain mitochondrial tubules and maintain mtDNA. Wild-type (FY833, WT), ugo1Δ (YHS72), dnm1Δ (YHS83), and ugo1Δ dnm1Δ (YHS85) cells expressing OM45-GFP (pKC2) were grown to log phase in SRaf medium, stained with 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP) microscopy. (B) Wild-type, ugo1Δ, dnm1Δ, and ugo1Δ dnm1Δ cells were grown to log phase in YEPD medium. Cells were collected and resuspended in YEPD medium to an OD600 of 2. Cells were then diluted in 10-fold increments, and 10 μl of each dilution was spotted onto YEPD and YEPGE media and incubated at 30°C for 2 and 6 d, respectively. N, nuclear DNA staining. Bar, 3 μm.
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Figure 5: ugo1Δ dnm1Δ cells contain mitochondrial tubules and maintain mtDNA. Wild-type (FY833, WT), ugo1Δ (YHS72), dnm1Δ (YHS83), and ugo1Δ dnm1Δ (YHS85) cells expressing OM45-GFP (pKC2) were grown to log phase in SRaf medium, stained with 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP) microscopy. (B) Wild-type, ugo1Δ, dnm1Δ, and ugo1Δ dnm1Δ cells were grown to log phase in YEPD medium. Cells were collected and resuspended in YEPD medium to an OD600 of 2. Cells were then diluted in 10-fold increments, and 10 μl of each dilution was spotted onto YEPD and YEPGE media and incubated at 30°C for 2 and 6 d, respectively. N, nuclear DNA staining. Bar, 3 μm.

Mentions: Mitochondrial fusion and division are normally balanced in cells, leading to the few tubular-shaped mitochondria seen in wild-type cells. The fragmentation of mitochondria in fzo1 results from continued division in the absence of fusion, and disruption of a gene required for division, DNM1, in fzo1 cells restores normal mitochondrial shape and number (Sesaki and Jensen 1999). To test if ugo1 shows a similar interplay with dnm1 as that seen with fzo1 and dnm1, we compared mitochondrial shape in either ugo1Δ mutants or dnm1Δ mutants to those in ugo1Δ dnm1Δ double mutants. In dnm1Δ mutants, a single mitochondria consisting of a network of interconnected tubules is seen, resulting from ongoing fusion in the absence of mitochondrial division (Fig. 5 A; Bleazard et al. 1999; Sesaki and Jensen 1999). As noted previously, ugo1Δ cells contain many small mitochondrial fragments (Fig. 3 and Fig. 5). In contrast, in the majority of ugo1Δ dnm1Δ cells (90%, n = 100), mitochondria appeared as elongated tubules, similar to those in wild-type cells (Fig. 5 A) or in fzo1 dnm1 double mutants (Sesaki and Jensen 1999). In ∼56% of ugo1Δ dnm1Δ cells mitochondrial tubules were often collapsed to one side of the cell and appeared to be bundled (Fig. 5 A, left panel of ugo1Δ dnm1Δ images). In 34% of ugo1Δ dnm1Δ cells individual mitochondrial tubules were clearly separated from other tubules (Fig. 5 A, right panel of ugo1Δ dnm1Δ images). Only a small fraction of ugo1Δ dnm1Δ cells (∼10%) showed mitochondria that appeared to be fragmented and aggregated. Thus, our results demonstrate that the fragmentation of mitochondria in ugo1Δ cells can be suppressed by dnm1 disruption. Ugo1p, like Fzo1p, appears to function in mitochondrial fusion, an activity antagonistic to the Dnm1p-mediated division of mitochondria.


UGO1 encodes an outer membrane protein required for mitochondrial fusion.

Sesaki H, Jensen RE - J. Cell Biol. (2001)

ugo1Δ dnm1Δ cells contain mitochondrial tubules and maintain mtDNA. Wild-type (FY833, WT), ugo1Δ (YHS72), dnm1Δ (YHS83), and ugo1Δ dnm1Δ (YHS85) cells expressing OM45-GFP (pKC2) were grown to log phase in SRaf medium, stained with 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP) microscopy. (B) Wild-type, ugo1Δ, dnm1Δ, and ugo1Δ dnm1Δ cells were grown to log phase in YEPD medium. Cells were collected and resuspended in YEPD medium to an OD600 of 2. Cells were then diluted in 10-fold increments, and 10 μl of each dilution was spotted onto YEPD and YEPGE media and incubated at 30°C for 2 and 6 d, respectively. N, nuclear DNA staining. Bar, 3 μm.
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Related In: Results  -  Collection

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Figure 5: ugo1Δ dnm1Δ cells contain mitochondrial tubules and maintain mtDNA. Wild-type (FY833, WT), ugo1Δ (YHS72), dnm1Δ (YHS83), and ugo1Δ dnm1Δ (YHS85) cells expressing OM45-GFP (pKC2) were grown to log phase in SRaf medium, stained with 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP) microscopy. (B) Wild-type, ugo1Δ, dnm1Δ, and ugo1Δ dnm1Δ cells were grown to log phase in YEPD medium. Cells were collected and resuspended in YEPD medium to an OD600 of 2. Cells were then diluted in 10-fold increments, and 10 μl of each dilution was spotted onto YEPD and YEPGE media and incubated at 30°C for 2 and 6 d, respectively. N, nuclear DNA staining. Bar, 3 μm.
Mentions: Mitochondrial fusion and division are normally balanced in cells, leading to the few tubular-shaped mitochondria seen in wild-type cells. The fragmentation of mitochondria in fzo1 results from continued division in the absence of fusion, and disruption of a gene required for division, DNM1, in fzo1 cells restores normal mitochondrial shape and number (Sesaki and Jensen 1999). To test if ugo1 shows a similar interplay with dnm1 as that seen with fzo1 and dnm1, we compared mitochondrial shape in either ugo1Δ mutants or dnm1Δ mutants to those in ugo1Δ dnm1Δ double mutants. In dnm1Δ mutants, a single mitochondria consisting of a network of interconnected tubules is seen, resulting from ongoing fusion in the absence of mitochondrial division (Fig. 5 A; Bleazard et al. 1999; Sesaki and Jensen 1999). As noted previously, ugo1Δ cells contain many small mitochondrial fragments (Fig. 3 and Fig. 5). In contrast, in the majority of ugo1Δ dnm1Δ cells (90%, n = 100), mitochondria appeared as elongated tubules, similar to those in wild-type cells (Fig. 5 A) or in fzo1 dnm1 double mutants (Sesaki and Jensen 1999). In ∼56% of ugo1Δ dnm1Δ cells mitochondrial tubules were often collapsed to one side of the cell and appeared to be bundled (Fig. 5 A, left panel of ugo1Δ dnm1Δ images). In 34% of ugo1Δ dnm1Δ cells individual mitochondrial tubules were clearly separated from other tubules (Fig. 5 A, right panel of ugo1Δ dnm1Δ images). Only a small fraction of ugo1Δ dnm1Δ cells (∼10%) showed mitochondria that appeared to be fragmented and aggregated. Thus, our results demonstrate that the fragmentation of mitochondria in ugo1Δ cells can be suppressed by dnm1 disruption. Ugo1p, like Fzo1p, appears to function in mitochondrial fusion, an activity antagonistic to the Dnm1p-mediated division of mitochondria.

Bottom Line: In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents.We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane.Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. hsesaki@jhmi.edu

ABSTRACT
Membrane fusion plays an important role in controlling the shape, number, and distribution of mitochondria. In the yeast Saccharomyces cerevisiae, the outer membrane protein Fzo1p has been shown to mediate mitochondrial fusion. Using a novel genetic screen, we have isolated new mutants defective in the fusion of their mitochondria. One of these mutants, ugo1, shows several similarities to fzo1 mutants. ugo1 cells contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. ugo1 mutants lose mitochondrial DNA (mtDNA). In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents. Fragmentation of mitochondria and loss of mtDNA in ugo1 mutants are rescued by disrupting DNM1, a gene required for mitochondrial division. We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane. Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space. Our results suggest that Ugo1p is a new outer membrane component of the mitochondrial fusion machinery.

Show MeSH
Related in: MedlinePlus