Limits...
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.

Show MeSH

Related in: MedlinePlus

Isolation of ugo mutants. (A) Strain used to isolate ugo1 and ugo2. ade2 DNM1 strains that carry plasmid pHS50, which expresses the dominant negative Dnm1-111 protein, lack Dnm1p activity. Cells that lose pHS50 contain functional Dnm1p. (B) Mutant isolation scheme. The parental strain (WT) maintains mtDNA on glucose-containing medium in the presence (SD-Ura) or absence (5FOAD) of the URA3-DNM1-111 plasmid and form red colonies due to the ade2 mutation (Reaume and Tatum 1949). ugo mutants maintain mtDNA only in the presence of the pHS50, forming red colonies on SD-Ura, but become white on 5FOAD medium, which selects for loss of the URA3-DNM1-111 plasmid (Boeke et al. 1984). Growth on glycerol and ethanol medium confirms that ugo mutants contain mtDNA in the presence of the URA3-DNM1-111 plasmid (YPGE), but that ugo mutants are inviable on 5FOA medium containing glycerol and ethanol (5FOAGE). (C) Four different genes were identified in the ugo screen. Complementation tests showed one fzo1 mutant, five mgm1 mutants, and two new mutants, ugo1 and ugo2, were isolated. (D) ugo mutants contain highly fragmented mitochondria. Wild-type (W303), ugo1-1 (YHS64), and ugo2-1 (YHS65) were grown on 5FOAD medium to select for cells that lost the URA3-DNM1-111 plasmid, pHS50, and then transformed with pKC2, which expresses GFP fused to the COOH terminus of the mitochondrial outer membrane protein, OM45p (OM45-GFP; Cerveny et al. 2001). Cells were grown to log phase in SRaf medium and examined by fluorescence microscopy. Fluorescence (OM45-GFP) and DIC images are shown. Bar, 3 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199209&req=5

Figure 1: Isolation of ugo mutants. (A) Strain used to isolate ugo1 and ugo2. ade2 DNM1 strains that carry plasmid pHS50, which expresses the dominant negative Dnm1-111 protein, lack Dnm1p activity. Cells that lose pHS50 contain functional Dnm1p. (B) Mutant isolation scheme. The parental strain (WT) maintains mtDNA on glucose-containing medium in the presence (SD-Ura) or absence (5FOAD) of the URA3-DNM1-111 plasmid and form red colonies due to the ade2 mutation (Reaume and Tatum 1949). ugo mutants maintain mtDNA only in the presence of the pHS50, forming red colonies on SD-Ura, but become white on 5FOAD medium, which selects for loss of the URA3-DNM1-111 plasmid (Boeke et al. 1984). Growth on glycerol and ethanol medium confirms that ugo mutants contain mtDNA in the presence of the URA3-DNM1-111 plasmid (YPGE), but that ugo mutants are inviable on 5FOA medium containing glycerol and ethanol (5FOAGE). (C) Four different genes were identified in the ugo screen. Complementation tests showed one fzo1 mutant, five mgm1 mutants, and two new mutants, ugo1 and ugo2, were isolated. (D) ugo mutants contain highly fragmented mitochondria. Wild-type (W303), ugo1-1 (YHS64), and ugo2-1 (YHS65) were grown on 5FOAD medium to select for cells that lost the URA3-DNM1-111 plasmid, pHS50, and then transformed with pKC2, which expresses GFP fused to the COOH terminus of the mitochondrial outer membrane protein, OM45p (OM45-GFP; Cerveny et al. 2001). Cells were grown to log phase in SRaf medium and examined by fluorescence microscopy. Fluorescence (OM45-GFP) and DIC images are shown. Bar, 3 μm.

Mentions: fzo1 mutants are defective in mitochondrial fusion and also lose mtDNA. The loss of mtDNA in fzo1 cells can be suppressed by inactivation of Dnm1p function (Bleazard et al. 1999; Sesaki and Jensen 1999; Jensen et al. 2000). To identify new components required for fusion, we screened for mutants that maintain mtDNA when Dnm1p activity is absent, but lose mtDNA in the presence of functional Dnm1p. We controlled Dnm1p activity using the URA3 plasmid pHS50, which carries a dominant negative version of Dnm1p, Dnm1p-111 (Fig. 1 A; Sesaki and Jensen 1999; Jensen et al. 2000). Cells that contain pHS50 lack Dnm1p function, which is restored upon loss of the plasmid. To monitor the presence of mtDNA, we took advantage of the ade2 mutation. ade2 cells that contain mtDNA are competent for respiration, producing a red pigment and forming red colonies (Reaume and Tatum 1949). Cells that lose mtDNA and the ability to respire form white colonies.


UGO1 encodes an outer membrane protein required for mitochondrial fusion.

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

Isolation of ugo mutants. (A) Strain used to isolate ugo1 and ugo2. ade2 DNM1 strains that carry plasmid pHS50, which expresses the dominant negative Dnm1-111 protein, lack Dnm1p activity. Cells that lose pHS50 contain functional Dnm1p. (B) Mutant isolation scheme. The parental strain (WT) maintains mtDNA on glucose-containing medium in the presence (SD-Ura) or absence (5FOAD) of the URA3-DNM1-111 plasmid and form red colonies due to the ade2 mutation (Reaume and Tatum 1949). ugo mutants maintain mtDNA only in the presence of the pHS50, forming red colonies on SD-Ura, but become white on 5FOAD medium, which selects for loss of the URA3-DNM1-111 plasmid (Boeke et al. 1984). Growth on glycerol and ethanol medium confirms that ugo mutants contain mtDNA in the presence of the URA3-DNM1-111 plasmid (YPGE), but that ugo mutants are inviable on 5FOA medium containing glycerol and ethanol (5FOAGE). (C) Four different genes were identified in the ugo screen. Complementation tests showed one fzo1 mutant, five mgm1 mutants, and two new mutants, ugo1 and ugo2, were isolated. (D) ugo mutants contain highly fragmented mitochondria. Wild-type (W303), ugo1-1 (YHS64), and ugo2-1 (YHS65) were grown on 5FOAD medium to select for cells that lost the URA3-DNM1-111 plasmid, pHS50, and then transformed with pKC2, which expresses GFP fused to the COOH terminus of the mitochondrial outer membrane protein, OM45p (OM45-GFP; Cerveny et al. 2001). Cells were grown to log phase in SRaf medium and examined by fluorescence microscopy. Fluorescence (OM45-GFP) and DIC images are shown. Bar, 3 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199209&req=5

Figure 1: Isolation of ugo mutants. (A) Strain used to isolate ugo1 and ugo2. ade2 DNM1 strains that carry plasmid pHS50, which expresses the dominant negative Dnm1-111 protein, lack Dnm1p activity. Cells that lose pHS50 contain functional Dnm1p. (B) Mutant isolation scheme. The parental strain (WT) maintains mtDNA on glucose-containing medium in the presence (SD-Ura) or absence (5FOAD) of the URA3-DNM1-111 plasmid and form red colonies due to the ade2 mutation (Reaume and Tatum 1949). ugo mutants maintain mtDNA only in the presence of the pHS50, forming red colonies on SD-Ura, but become white on 5FOAD medium, which selects for loss of the URA3-DNM1-111 plasmid (Boeke et al. 1984). Growth on glycerol and ethanol medium confirms that ugo mutants contain mtDNA in the presence of the URA3-DNM1-111 plasmid (YPGE), but that ugo mutants are inviable on 5FOA medium containing glycerol and ethanol (5FOAGE). (C) Four different genes were identified in the ugo screen. Complementation tests showed one fzo1 mutant, five mgm1 mutants, and two new mutants, ugo1 and ugo2, were isolated. (D) ugo mutants contain highly fragmented mitochondria. Wild-type (W303), ugo1-1 (YHS64), and ugo2-1 (YHS65) were grown on 5FOAD medium to select for cells that lost the URA3-DNM1-111 plasmid, pHS50, and then transformed with pKC2, which expresses GFP fused to the COOH terminus of the mitochondrial outer membrane protein, OM45p (OM45-GFP; Cerveny et al. 2001). Cells were grown to log phase in SRaf medium and examined by fluorescence microscopy. Fluorescence (OM45-GFP) and DIC images are shown. Bar, 3 μm.
Mentions: fzo1 mutants are defective in mitochondrial fusion and also lose mtDNA. The loss of mtDNA in fzo1 cells can be suppressed by inactivation of Dnm1p function (Bleazard et al. 1999; Sesaki and Jensen 1999; Jensen et al. 2000). To identify new components required for fusion, we screened for mutants that maintain mtDNA when Dnm1p activity is absent, but lose mtDNA in the presence of functional Dnm1p. We controlled Dnm1p activity using the URA3 plasmid pHS50, which carries a dominant negative version of Dnm1p, Dnm1p-111 (Fig. 1 A; Sesaki and Jensen 1999; Jensen et al. 2000). Cells that contain pHS50 lack Dnm1p function, which is restored upon loss of the plasmid. To monitor the presence of mtDNA, we took advantage of the ade2 mutation. ade2 cells that contain mtDNA are competent for respiration, producing a red pigment and forming red colonies (Reaume and Tatum 1949). Cells that lose mtDNA and the ability to respire form white colonies.

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