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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 located in the mitochondrial outer membrane, with its NH2 terminus facing the cytosol and COOH terminus in the IMS. (A) Ugo1p is an integral membrane protein. Mitochondria isolated from ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were treated with either 1.5 M sodium chloride, 0.1 M sodium carbonate, or untreated (Control). Mitochondrial membranes were then separated into supernatant (S) and pellet (P) fractions by centrifugation at 100,000 g for 60 min. Aliquots from each fraction were analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), Tim23p, an integral membrane protein, and the β subunit of the F1-ATPase (F1β), a peripheral membrane protein. (B) Ugo1p is located in the outer membrane. myc-Ugo1p mitochondria were sonicated and membrane vesicles were loaded on sucrose gradients. After centrifugation, fractions were collected and analyzed by immune blotting with antibodies to the myc epitope (myc-Ugo1p), the outer membrane protein OM45, and the inner membrane protein F1β. Fraction 1 represents the top of the gradient. (C) The COOH terminus of Ugo1p faces the IMS. Ugo1p-HA mitochondria were digested with 200 μg/ml trypsin for 20 min on ice and analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), OM45, and the inner membrane proteins, F1β and Tim23p. To expose proteins located in the IMS, the mitochondrial outer membrane was disrupted by osmotic shock (OS) and then treated with protease. Asterisk, a proteolytic fragment of the COOH terminus of Ugo1p-HA. (D) The NH2 terminus of Ugo1p faces the cytosol. Mitochondria were isolated from ugo1Δ cells (YHS72) expressing myc-Ugo1p (pHS57). Mitochondria were treated with trypsin and analyzed by immune blotting.
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Figure 8: Ugo1p is located in the mitochondrial outer membrane, with its NH2 terminus facing the cytosol and COOH terminus in the IMS. (A) Ugo1p is an integral membrane protein. Mitochondria isolated from ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were treated with either 1.5 M sodium chloride, 0.1 M sodium carbonate, or untreated (Control). Mitochondrial membranes were then separated into supernatant (S) and pellet (P) fractions by centrifugation at 100,000 g for 60 min. Aliquots from each fraction were analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), Tim23p, an integral membrane protein, and the β subunit of the F1-ATPase (F1β), a peripheral membrane protein. (B) Ugo1p is located in the outer membrane. myc-Ugo1p mitochondria were sonicated and membrane vesicles were loaded on sucrose gradients. After centrifugation, fractions were collected and analyzed by immune blotting with antibodies to the myc epitope (myc-Ugo1p), the outer membrane protein OM45, and the inner membrane protein F1β. Fraction 1 represents the top of the gradient. (C) The COOH terminus of Ugo1p faces the IMS. Ugo1p-HA mitochondria were digested with 200 μg/ml trypsin for 20 min on ice and analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), OM45, and the inner membrane proteins, F1β and Tim23p. To expose proteins located in the IMS, the mitochondrial outer membrane was disrupted by osmotic shock (OS) and then treated with protease. Asterisk, a proteolytic fragment of the COOH terminus of Ugo1p-HA. (D) The NH2 terminus of Ugo1p faces the cytosol. Mitochondria were isolated from ugo1Δ cells (YHS72) expressing myc-Ugo1p (pHS57). Mitochondria were treated with trypsin and analyzed by immune blotting.

Mentions: Ugo1p is an integral membrane protein located in the outer membrane. When mitochondria isolated from cells expressing Ugo1p-HA were treated with 1.5 M sodium chloride or 0.1 M sodium carbonate (Fig. 8 A), Ugo1p was not extracted from the mitochondria like the peripheral membrane protein, the β subunit of the F1-ATPase (F1β). Instead, Ugo1p remained in the membrane pellet with the integral membrane protein, Tim23p. To determine which mitochondrial membrane contains Ugo1p, we prepared membrane vesicles from myc-Ugo1p mitochondria and separated them into outer membrane and inner membrane fractions on sucrose gradients. As shown in Fig. 8 B, myc-Ugo1 cofractionated with the outer membrane vesicle fraction, along with OM45. The F1β protein, a marker for the inner membrane, was found in more dense fractions, separate from myc-Ugo1p and OM45.


UGO1 encodes an outer membrane protein required for mitochondrial fusion.

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

Ugo1p is located in the mitochondrial outer membrane, with its NH2 terminus facing the cytosol and COOH terminus in the IMS. (A) Ugo1p is an integral membrane protein. Mitochondria isolated from ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were treated with either 1.5 M sodium chloride, 0.1 M sodium carbonate, or untreated (Control). Mitochondrial membranes were then separated into supernatant (S) and pellet (P) fractions by centrifugation at 100,000 g for 60 min. Aliquots from each fraction were analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), Tim23p, an integral membrane protein, and the β subunit of the F1-ATPase (F1β), a peripheral membrane protein. (B) Ugo1p is located in the outer membrane. myc-Ugo1p mitochondria were sonicated and membrane vesicles were loaded on sucrose gradients. After centrifugation, fractions were collected and analyzed by immune blotting with antibodies to the myc epitope (myc-Ugo1p), the outer membrane protein OM45, and the inner membrane protein F1β. Fraction 1 represents the top of the gradient. (C) The COOH terminus of Ugo1p faces the IMS. Ugo1p-HA mitochondria were digested with 200 μg/ml trypsin for 20 min on ice and analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), OM45, and the inner membrane proteins, F1β and Tim23p. To expose proteins located in the IMS, the mitochondrial outer membrane was disrupted by osmotic shock (OS) and then treated with protease. Asterisk, a proteolytic fragment of the COOH terminus of Ugo1p-HA. (D) The NH2 terminus of Ugo1p faces the cytosol. Mitochondria were isolated from ugo1Δ cells (YHS72) expressing myc-Ugo1p (pHS57). Mitochondria were treated with trypsin and analyzed by immune blotting.
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Figure 8: Ugo1p is located in the mitochondrial outer membrane, with its NH2 terminus facing the cytosol and COOH terminus in the IMS. (A) Ugo1p is an integral membrane protein. Mitochondria isolated from ugo1Δ cells (YHS87) expressing Ugo1p-HA (pHS55) were treated with either 1.5 M sodium chloride, 0.1 M sodium carbonate, or untreated (Control). Mitochondrial membranes were then separated into supernatant (S) and pellet (P) fractions by centrifugation at 100,000 g for 60 min. Aliquots from each fraction were analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), Tim23p, an integral membrane protein, and the β subunit of the F1-ATPase (F1β), a peripheral membrane protein. (B) Ugo1p is located in the outer membrane. myc-Ugo1p mitochondria were sonicated and membrane vesicles were loaded on sucrose gradients. After centrifugation, fractions were collected and analyzed by immune blotting with antibodies to the myc epitope (myc-Ugo1p), the outer membrane protein OM45, and the inner membrane protein F1β. Fraction 1 represents the top of the gradient. (C) The COOH terminus of Ugo1p faces the IMS. Ugo1p-HA mitochondria were digested with 200 μg/ml trypsin for 20 min on ice and analyzed by immune blotting with antibodies to the HA epitope (Ugo1p-HA), OM45, and the inner membrane proteins, F1β and Tim23p. To expose proteins located in the IMS, the mitochondrial outer membrane was disrupted by osmotic shock (OS) and then treated with protease. Asterisk, a proteolytic fragment of the COOH terminus of Ugo1p-HA. (D) The NH2 terminus of Ugo1p faces the cytosol. Mitochondria were isolated from ugo1Δ cells (YHS72) expressing myc-Ugo1p (pHS57). Mitochondria were treated with trypsin and analyzed by immune blotting.
Mentions: Ugo1p is an integral membrane protein located in the outer membrane. When mitochondria isolated from cells expressing Ugo1p-HA were treated with 1.5 M sodium chloride or 0.1 M sodium carbonate (Fig. 8 A), Ugo1p was not extracted from the mitochondria like the peripheral membrane protein, the β subunit of the F1-ATPase (F1β). Instead, Ugo1p remained in the membrane pellet with the integral membrane protein, Tim23p. To determine which mitochondrial membrane contains Ugo1p, we prepared membrane vesicles from myc-Ugo1p mitochondria and separated them into outer membrane and inner membrane fractions on sucrose gradients. As shown in Fig. 8 B, myc-Ugo1 cofractionated with the outer membrane vesicle fraction, along with OM45. The F1β protein, a marker for the inner membrane, was found in more dense fractions, separate from myc-Ugo1p and OM45.

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