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Mitochondrial outer and inner membrane fusion requires a modified carrier protein.

Hoppins S, Horner J, Song C, McCaffery JM, Nunnari J - J. Cell Biol. (2009)

Bottom Line: Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively.At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing.The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.

ABSTRACT
In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

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Native Ugo1 forms a 115-kD homooligomer that requires the conserved ETMs. (A) Mitochondria isolated from Δugo1 plus empty vector or Δugo1 expressing either UGO1, Ugo1D134R, Ugo1R137D, Ugo1D312R, or Ugo1R315D were solubilized in 1.5% digitonin and analyzed by hrCN-PAGE and immunodetection with Ugo1 polyclonal antiserum. Small amounts of nonspecific bands detected by the sera are visible on the blots (single arrows) in addition to the Ugo1 species (double arrow). (B) Immunoprecipitations were performed with either α-HA monoclonal antibodies (left) or α-Flag monoclonal antibodies (right) on 125 µg chemically cross-linked mitochondria isolated from a UGO1-Flag strain or UGO1-Flag strain expressing Ugo1-HA. Proteins were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Total (T) represents 2.5% of the input, and pellet (P) represents 25% of the immunoprecipitated protein. (C) To examine protein stability, the digitonin-solubilized mitochondria in A (10 µg) were separated by SDS-PAGE and visualized by Western analysis with α-Ugo1 or α-porin. The graph represents the quantification of digitonin lysate Ugo1 total protein level normalized to porin as detected by SDS-PAGE analysis versus that detected in the 115-kD Ugo1 species resolved by hrCN-PAGE analysis. Quantification was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences).
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fig3: Native Ugo1 forms a 115-kD homooligomer that requires the conserved ETMs. (A) Mitochondria isolated from Δugo1 plus empty vector or Δugo1 expressing either UGO1, Ugo1D134R, Ugo1R137D, Ugo1D312R, or Ugo1R315D were solubilized in 1.5% digitonin and analyzed by hrCN-PAGE and immunodetection with Ugo1 polyclonal antiserum. Small amounts of nonspecific bands detected by the sera are visible on the blots (single arrows) in addition to the Ugo1 species (double arrow). (B) Immunoprecipitations were performed with either α-HA monoclonal antibodies (left) or α-Flag monoclonal antibodies (right) on 125 µg chemically cross-linked mitochondria isolated from a UGO1-Flag strain or UGO1-Flag strain expressing Ugo1-HA. Proteins were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Total (T) represents 2.5% of the input, and pellet (P) represents 25% of the immunoprecipitated protein. (C) To examine protein stability, the digitonin-solubilized mitochondria in A (10 µg) were separated by SDS-PAGE and visualized by Western analysis with α-Ugo1 or α-porin. The graph represents the quantification of digitonin lysate Ugo1 total protein level normalized to porin as detected by SDS-PAGE analysis versus that detected in the 115-kD Ugo1 species resolved by hrCN-PAGE analysis. Quantification was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Mentions: To test this possibility, we focused our analysis on the quaternary structure of Ugo1. Specifically, we determined the native size of Ugo1 in yeast mitochondria by high resolution clear native PAGE (hrCN-PAGE; Wittig et al., 2007). After solubilization with digitonin, mitochondrial proteins were subjected to hrCN-PAGE that was capable of resolving 70–700-kD-sized protein species. By Western analysis with polyclonal antibodies directed against the N terminus of Ugo1, we observed that the most abundant form of Ugo1 in wild-type mitochondria was a species with an apparent molecular mass of 115 kD, which is consistent with the size of a Ugo1 dimer (Ugo1 Mr 57,470; Fig. 3 A, lane 1). This species was not detected with antibodies directed against either Fzo1 or Mgm1 and was present in mitochondria lacking Fzo1 or Mgm1 but was absent in mitochondria isolated from a Δugo1 strain, which is consistent with the interpretation that this 115-kD species represents a Ugo1 homodimer (Fig. 3 A, lane 2; and not depicted). To further test that Ugo1 exists as dimer, we coexpressed Ugo1-HA and Ugo1-Flag in cells and asked whether they interact in mitochondria by immunoprecipitation analysis. We observed that anti-HA antibodies immunoprecipitated Ugo1-HA and Ugo1-Flag from chemically cross-linked mitochondrial extracts as detected by Western analysis (Fig. 3 B). This interaction was specific as judged by the fact that anti-HA immunoprecipitation of Ugo1-Flag was dependent on the HA-tagged form of Ugo1 (Fig. 3 B, compare lane 2 with lane 4). In addition, analysis of the reciprocal immunoprecipitation using anti-Flag antibodies detected a Ugo1-Flag–Ugo1-HA interaction, further supporting our conclusion that Ugo1 forms a dimer in the mitochondrial outer membrane (Fig. 3 B, lane 6).


Mitochondrial outer and inner membrane fusion requires a modified carrier protein.

Hoppins S, Horner J, Song C, McCaffery JM, Nunnari J - J. Cell Biol. (2009)

Native Ugo1 forms a 115-kD homooligomer that requires the conserved ETMs. (A) Mitochondria isolated from Δugo1 plus empty vector or Δugo1 expressing either UGO1, Ugo1D134R, Ugo1R137D, Ugo1D312R, or Ugo1R315D were solubilized in 1.5% digitonin and analyzed by hrCN-PAGE and immunodetection with Ugo1 polyclonal antiserum. Small amounts of nonspecific bands detected by the sera are visible on the blots (single arrows) in addition to the Ugo1 species (double arrow). (B) Immunoprecipitations were performed with either α-HA monoclonal antibodies (left) or α-Flag monoclonal antibodies (right) on 125 µg chemically cross-linked mitochondria isolated from a UGO1-Flag strain or UGO1-Flag strain expressing Ugo1-HA. Proteins were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Total (T) represents 2.5% of the input, and pellet (P) represents 25% of the immunoprecipitated protein. (C) To examine protein stability, the digitonin-solubilized mitochondria in A (10 µg) were separated by SDS-PAGE and visualized by Western analysis with α-Ugo1 or α-porin. The graph represents the quantification of digitonin lysate Ugo1 total protein level normalized to porin as detected by SDS-PAGE analysis versus that detected in the 115-kD Ugo1 species resolved by hrCN-PAGE analysis. Quantification was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654124&req=5

fig3: Native Ugo1 forms a 115-kD homooligomer that requires the conserved ETMs. (A) Mitochondria isolated from Δugo1 plus empty vector or Δugo1 expressing either UGO1, Ugo1D134R, Ugo1R137D, Ugo1D312R, or Ugo1R315D were solubilized in 1.5% digitonin and analyzed by hrCN-PAGE and immunodetection with Ugo1 polyclonal antiserum. Small amounts of nonspecific bands detected by the sera are visible on the blots (single arrows) in addition to the Ugo1 species (double arrow). (B) Immunoprecipitations were performed with either α-HA monoclonal antibodies (left) or α-Flag monoclonal antibodies (right) on 125 µg chemically cross-linked mitochondria isolated from a UGO1-Flag strain or UGO1-Flag strain expressing Ugo1-HA. Proteins were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Total (T) represents 2.5% of the input, and pellet (P) represents 25% of the immunoprecipitated protein. (C) To examine protein stability, the digitonin-solubilized mitochondria in A (10 µg) were separated by SDS-PAGE and visualized by Western analysis with α-Ugo1 or α-porin. The graph represents the quantification of digitonin lysate Ugo1 total protein level normalized to porin as detected by SDS-PAGE analysis versus that detected in the 115-kD Ugo1 species resolved by hrCN-PAGE analysis. Quantification was performed using the Odyssey Infrared Imaging System (LI-COR Biosciences).
Mentions: To test this possibility, we focused our analysis on the quaternary structure of Ugo1. Specifically, we determined the native size of Ugo1 in yeast mitochondria by high resolution clear native PAGE (hrCN-PAGE; Wittig et al., 2007). After solubilization with digitonin, mitochondrial proteins were subjected to hrCN-PAGE that was capable of resolving 70–700-kD-sized protein species. By Western analysis with polyclonal antibodies directed against the N terminus of Ugo1, we observed that the most abundant form of Ugo1 in wild-type mitochondria was a species with an apparent molecular mass of 115 kD, which is consistent with the size of a Ugo1 dimer (Ugo1 Mr 57,470; Fig. 3 A, lane 1). This species was not detected with antibodies directed against either Fzo1 or Mgm1 and was present in mitochondria lacking Fzo1 or Mgm1 but was absent in mitochondria isolated from a Δugo1 strain, which is consistent with the interpretation that this 115-kD species represents a Ugo1 homodimer (Fig. 3 A, lane 2; and not depicted). To further test that Ugo1 exists as dimer, we coexpressed Ugo1-HA and Ugo1-Flag in cells and asked whether they interact in mitochondria by immunoprecipitation analysis. We observed that anti-HA antibodies immunoprecipitated Ugo1-HA and Ugo1-Flag from chemically cross-linked mitochondrial extracts as detected by Western analysis (Fig. 3 B). This interaction was specific as judged by the fact that anti-HA immunoprecipitation of Ugo1-Flag was dependent on the HA-tagged form of Ugo1 (Fig. 3 B, compare lane 2 with lane 4). In addition, analysis of the reciprocal immunoprecipitation using anti-Flag antibodies detected a Ugo1-Flag–Ugo1-HA interaction, further supporting our conclusion that Ugo1 forms a dimer in the mitochondrial outer membrane (Fig. 3 B, lane 6).

Bottom Line: Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively.At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing.The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.

ABSTRACT
In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

Show MeSH
Related in: MedlinePlus