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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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Ugo1p is a mitochondrial protein. (A) Expression of myc-Ugo1p. Wild-type cells (FY833) containing an empty vector, pRS314 (Control), and ugo1Δ cells (YHS88) containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Whole cell extracts were prepared (Yaffe and Schatz 1984) and analyzed by immune blotting using antibodies to the myc epitope. (B) Ugo1p colocalizes with a mitochondrial protein. Wild-type cells containing pRS314 (Ugo1p) and ugo1Δ cells containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Cells were then fixed, spheroplasted, permeabilized (Harlow and Lane 1988), and incubated with rabbit antibodies to the β subunit of F1-ATPase (anti-F1β) and mouse IgG to the myc epitope (anti-myc). Immune complexes were visualized by fluorescence microscopy using FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rabbit IgG. (C) Ugo1p cofractionates with a mitochondrial marker. ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were grown in SGal medium. Cells were homogenized and separated into a mitochondrial pellet and a postmitochondrial supernatant by centrifugation. Cell-equivalent amounts of homogenate (H), mitochondrial pellet (M), and postmitochondrial supernatant (PMS) were analyzed by immune blotting using antibodies to the HA epitope (Ugo1p-HA), Tim23p, and hexokinase. Bar, 3 μm.
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Figure 7: Ugo1p is a mitochondrial protein. (A) Expression of myc-Ugo1p. Wild-type cells (FY833) containing an empty vector, pRS314 (Control), and ugo1Δ cells (YHS88) containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Whole cell extracts were prepared (Yaffe and Schatz 1984) and analyzed by immune blotting using antibodies to the myc epitope. (B) Ugo1p colocalizes with a mitochondrial protein. Wild-type cells containing pRS314 (Ugo1p) and ugo1Δ cells containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Cells were then fixed, spheroplasted, permeabilized (Harlow and Lane 1988), and incubated with rabbit antibodies to the β subunit of F1-ATPase (anti-F1β) and mouse IgG to the myc epitope (anti-myc). Immune complexes were visualized by fluorescence microscopy using FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rabbit IgG. (C) Ugo1p cofractionates with a mitochondrial marker. ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were grown in SGal medium. Cells were homogenized and separated into a mitochondrial pellet and a postmitochondrial supernatant by centrifugation. Cell-equivalent amounts of homogenate (H), mitochondrial pellet (M), and postmitochondrial supernatant (PMS) were analyzed by immune blotting using antibodies to the HA epitope (Ugo1p-HA), Tim23p, and hexokinase. Bar, 3 μm.

Mentions: To localize Ugo1p in yeast cells, we constructed two epitope-tagged versions of Ugo1p, myc-Ugo1p, and Ugo1p-HA. myc-Ugo1p carries the myc epitope (Munro and Pelham 1986) fused to the NH2 terminus of the Ugo1 protein, and Ugo1p-HA contains the influenza HA epitope (Field et al. 1988) at its COOH terminus. Cells that expressed either myc-Ugo1p (Fig. 7 A) or Ugo1p-HA (data not shown) contained a single protein of ∼65 kD. We found that both fusion proteins were functional and ugo1Δ cells expressing either myc-Ugo1p (Fig. 7 B) or Ugo1p-HA (data not shown) maintained mtDNA and normal mitochondrial shape.


UGO1 encodes an outer membrane protein required for mitochondrial fusion.

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

Ugo1p is a mitochondrial protein. (A) Expression of myc-Ugo1p. Wild-type cells (FY833) containing an empty vector, pRS314 (Control), and ugo1Δ cells (YHS88) containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Whole cell extracts were prepared (Yaffe and Schatz 1984) and analyzed by immune blotting using antibodies to the myc epitope. (B) Ugo1p colocalizes with a mitochondrial protein. Wild-type cells containing pRS314 (Ugo1p) and ugo1Δ cells containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Cells were then fixed, spheroplasted, permeabilized (Harlow and Lane 1988), and incubated with rabbit antibodies to the β subunit of F1-ATPase (anti-F1β) and mouse IgG to the myc epitope (anti-myc). Immune complexes were visualized by fluorescence microscopy using FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rabbit IgG. (C) Ugo1p cofractionates with a mitochondrial marker. ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were grown in SGal medium. Cells were homogenized and separated into a mitochondrial pellet and a postmitochondrial supernatant by centrifugation. Cell-equivalent amounts of homogenate (H), mitochondrial pellet (M), and postmitochondrial supernatant (PMS) were analyzed by immune blotting using antibodies to the HA epitope (Ugo1p-HA), Tim23p, and hexokinase. Bar, 3 μm.
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Figure 7: Ugo1p is a mitochondrial protein. (A) Expression of myc-Ugo1p. Wild-type cells (FY833) containing an empty vector, pRS314 (Control), and ugo1Δ cells (YHS88) containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Whole cell extracts were prepared (Yaffe and Schatz 1984) and analyzed by immune blotting using antibodies to the myc epitope. (B) Ugo1p colocalizes with a mitochondrial protein. Wild-type cells containing pRS314 (Ugo1p) and ugo1Δ cells containing the myc-Ugo1p plasmid (pHS57) were grown to log phase in SGal medium. Cells were then fixed, spheroplasted, permeabilized (Harlow and Lane 1988), and incubated with rabbit antibodies to the β subunit of F1-ATPase (anti-F1β) and mouse IgG to the myc epitope (anti-myc). Immune complexes were visualized by fluorescence microscopy using FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rabbit IgG. (C) Ugo1p cofractionates with a mitochondrial marker. ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were grown in SGal medium. Cells were homogenized and separated into a mitochondrial pellet and a postmitochondrial supernatant by centrifugation. Cell-equivalent amounts of homogenate (H), mitochondrial pellet (M), and postmitochondrial supernatant (PMS) were analyzed by immune blotting using antibodies to the HA epitope (Ugo1p-HA), Tim23p, and hexokinase. Bar, 3 μm.
Mentions: To localize Ugo1p in yeast cells, we constructed two epitope-tagged versions of Ugo1p, myc-Ugo1p, and Ugo1p-HA. myc-Ugo1p carries the myc epitope (Munro and Pelham 1986) fused to the NH2 terminus of the Ugo1 protein, and Ugo1p-HA contains the influenza HA epitope (Field et al. 1988) at its COOH terminus. Cells that expressed either myc-Ugo1p (Fig. 7 A) or Ugo1p-HA (data not shown) contained a single protein of ∼65 kD. We found that both fusion proteins were functional and ugo1Δ cells expressing either myc-Ugo1p (Fig. 7 B) or Ugo1p-HA (data not shown) maintained mtDNA and normal mitochondrial shape.

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