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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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Related in: MedlinePlus

Ugo1 contains three membrane-spanning domains. (A) A schematic of Ugo1 as in Fig. 1 with arrows indicating the locations of internal triple HA tags. (B) Protease protection analysis of internal HA-tagged versions of Ugo1, which are schematically depicted in A. The protected C-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the antibody with trypsin (IRdye-α rabbit). (C) Model of Ugo1 topology in the outer mitochondrial membrane. The location of the putative carrier domains are each indicated with an asterisk. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.
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fig2: Ugo1 contains three membrane-spanning domains. (A) A schematic of Ugo1 as in Fig. 1 with arrows indicating the locations of internal triple HA tags. (B) Protease protection analysis of internal HA-tagged versions of Ugo1, which are schematically depicted in A. The protected C-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the antibody with trypsin (IRdye-α rabbit). (C) Model of Ugo1 topology in the outer mitochondrial membrane. The location of the putative carrier domains are each indicated with an asterisk. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.

Mentions: To distinguish between these models, we generated versions of Ugo1 with internal HA tags located in predicted loop regions that follow potential TMDs ending at amino acid 393 (Ugo1HA393) or 438 (Ugo1HA438; Fig. 2 A). Both Ugo1HA393 and Ugo1HA438 were functional as assessed by their ability to rescue the mitochondrial DNA loss and glycerol growth defects of Δugo1 cells (unpublished data), indicating that they are correctly targeted to the outer membrane and are suitable for topology analysis. Protease protection analysis of intact mitochondria isolated from yeast strains expressing Ugo1HA393 and Ugo1HA438 revealed that the HA epitope in both versions is protease resistant but becomes sensitive to trypsin proteolysis in outer membrane–permeabilized mitoplasts (Fig. 2 B, lanes 1, 3, and 5). These data indicate that regions of Ugo1 at amino acid 393 and 438 are localized to the IMS. Consistently, these regions were readily proteolyzed in detergent-solubilized mitochondria and were not detected in the supernatant of the mitoplast fractions, indicating that they are protected from proteolysis by an intact outer membrane (Fig. 2 B, lanes 2 and 4). We conclude from these data that there are no TMDs in the C-terminal half of Ugo1.


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)

Ugo1 contains three membrane-spanning domains. (A) A schematic of Ugo1 as in Fig. 1 with arrows indicating the locations of internal triple HA tags. (B) Protease protection analysis of internal HA-tagged versions of Ugo1, which are schematically depicted in A. The protected C-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the antibody with trypsin (IRdye-α rabbit). (C) Model of Ugo1 topology in the outer mitochondrial membrane. The location of the putative carrier domains are each indicated with an asterisk. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Ugo1 contains three membrane-spanning domains. (A) A schematic of Ugo1 as in Fig. 1 with arrows indicating the locations of internal triple HA tags. (B) Protease protection analysis of internal HA-tagged versions of Ugo1, which are schematically depicted in A. The protected C-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the antibody with trypsin (IRdye-α rabbit). (C) Model of Ugo1 topology in the outer mitochondrial membrane. The location of the putative carrier domains are each indicated with an asterisk. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.
Mentions: To distinguish between these models, we generated versions of Ugo1 with internal HA tags located in predicted loop regions that follow potential TMDs ending at amino acid 393 (Ugo1HA393) or 438 (Ugo1HA438; Fig. 2 A). Both Ugo1HA393 and Ugo1HA438 were functional as assessed by their ability to rescue the mitochondrial DNA loss and glycerol growth defects of Δugo1 cells (unpublished data), indicating that they are correctly targeted to the outer membrane and are suitable for topology analysis. Protease protection analysis of intact mitochondria isolated from yeast strains expressing Ugo1HA393 and Ugo1HA438 revealed that the HA epitope in both versions is protease resistant but becomes sensitive to trypsin proteolysis in outer membrane–permeabilized mitoplasts (Fig. 2 B, lanes 1, 3, and 5). These data indicate that regions of Ugo1 at amino acid 393 and 438 are localized to the IMS. Consistently, these regions were readily proteolyzed in detergent-solubilized mitochondria and were not detected in the supernatant of the mitoplast fractions, indicating that they are protected from proteolysis by an intact outer membrane (Fig. 2 B, lanes 2 and 4). We conclude from these data that there are no TMDs in the C-terminal half of Ugo1.

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