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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 cells contain fragmented mitochondria and lack mtDNA. Wild-type (FY833), rho0 WT (YHS92), and ugo1Δ (YHS72) and fzo1Δ (YHS74) cells were transformed with OM45-GFP–expressing plasmid, pKC2 (Cerveny et al. 2001). Cells were grown to log phase in SRaf medium. Cells were stained using 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP or DAPI) microscopy. rho0 cells (YHS92) were generated by treating wild-type cells (FY833) with 25 μg/ml ethidium bromide as described (Fox et al. 1991). N, nuclear DNA staining. Bar, 3 μm.
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Figure 3: ugo1Δ cells contain fragmented mitochondria and lack mtDNA. Wild-type (FY833), rho0 WT (YHS92), and ugo1Δ (YHS72) and fzo1Δ (YHS74) cells were transformed with OM45-GFP–expressing plasmid, pKC2 (Cerveny et al. 2001). Cells were grown to log phase in SRaf medium. Cells were stained using 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP or DAPI) microscopy. rho0 cells (YHS92) were generated by treating wild-type cells (FY833) with 25 μg/ml ethidium bromide as described (Fox et al. 1991). N, nuclear DNA staining. Bar, 3 μm.

Mentions: To further investigate the function of Ugo1p, we created a allele by replacing the UGO1 open reading frame with the yeast HIS3 gene (ugo1Δ). UGO1/ugo1Δ diploid cells were sporulated and the haploid segregants were allowed to grow on YEPD. We found that all four spores in each tetrad were viable, but that two spores formed small colonies (Fig. 2 A). The slower growing cells were shown to carry the ugo1Δ gene, whereas cells from larger-sized colonies contained UGO1. Ugo1p appears to be required for normal cell growth. As shown in Fig. 2 B, cells carrying the ugo1Δ disruption failed to grow on YEPGE. Since mtDNA is essential for growth on glycerol and ethanol, we asked if ugo1Δ cells lack mtDNA by staining them with the DNA-specific dye, DAPI. DAPI staining showed that mtDNA nucleoids were present in wild-type cells (Fig. 3). In contrast, ugo1Δ cells contained little or no mtDNA and only faint staining of nuclear DNA was seen, similar to wild-type cells lacking mtDNA (rho0 WT). Our results indicate that UGO1 is required for growth on nonfermentable carbon sources and to maintain mtDNA.


UGO1 encodes an outer membrane protein required for mitochondrial fusion.

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

ugo1Δ cells contain fragmented mitochondria and lack mtDNA. Wild-type (FY833), rho0 WT (YHS92), and ugo1Δ (YHS72) and fzo1Δ (YHS74) cells were transformed with OM45-GFP–expressing plasmid, pKC2 (Cerveny et al. 2001). Cells were grown to log phase in SRaf medium. Cells were stained using 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP or DAPI) microscopy. rho0 cells (YHS92) were generated by treating wild-type cells (FY833) with 25 μg/ml ethidium bromide as described (Fox et al. 1991). N, nuclear DNA staining. Bar, 3 μm.
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Related In: Results  -  Collection

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Figure 3: ugo1Δ cells contain fragmented mitochondria and lack mtDNA. Wild-type (FY833), rho0 WT (YHS92), and ugo1Δ (YHS72) and fzo1Δ (YHS74) cells were transformed with OM45-GFP–expressing plasmid, pKC2 (Cerveny et al. 2001). Cells were grown to log phase in SRaf medium. Cells were stained using 1 μg/ml DAPI, and viewed by DIC and fluorescence (OM45-GFP or DAPI) microscopy. rho0 cells (YHS92) were generated by treating wild-type cells (FY833) with 25 μg/ml ethidium bromide as described (Fox et al. 1991). N, nuclear DNA staining. Bar, 3 μm.
Mentions: To further investigate the function of Ugo1p, we created a allele by replacing the UGO1 open reading frame with the yeast HIS3 gene (ugo1Δ). UGO1/ugo1Δ diploid cells were sporulated and the haploid segregants were allowed to grow on YEPD. We found that all four spores in each tetrad were viable, but that two spores formed small colonies (Fig. 2 A). The slower growing cells were shown to carry the ugo1Δ gene, whereas cells from larger-sized colonies contained UGO1. Ugo1p appears to be required for normal cell growth. As shown in Fig. 2 B, cells carrying the ugo1Δ disruption failed to grow on YEPGE. Since mtDNA is essential for growth on glycerol and ethanol, we asked if ugo1Δ cells lack mtDNA by staining them with the DNA-specific dye, DAPI. DAPI staining showed that mtDNA nucleoids were present in wild-type cells (Fig. 3). In contrast, ugo1Δ cells contained little or no mtDNA and only faint staining of nuclear DNA was seen, similar to wild-type cells lacking mtDNA (rho0 WT). Our results indicate that UGO1 is required for growth on nonfermentable carbon sources and to maintain mtDNA.

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