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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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Analysis of Ugo1 topology. (A and B) Mitochondria were left intact (lanes 5 and 10), converted to mitoplasts (lanes 3 and 8; mitoplast supernatant, lanes 4 and 9), or solubilized (lanes 2 and 7) before treatment with (+) or without (−) trypsin (lanes 1 and 6) and were analyzed by SDS-PAGE and immunoblotting with the indicated antisera. The 23-kD protected C-terminal Ugo1 fragment is indicated by a single arrow, and the 17–20-kD protected N-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the secondary antibody with trypsin (HRP-α rabbit). (C) Schematic of Ugo1 structure. Black boxes designate hydrophobic regions predicted to be bona fide TMDs based on data in A, and gray boxes represent additional possible TMDs. Amino acid positions are listed above the boxes. Predicted cytosolic (cyt) and IMS regions are also indicated. (D) Schematic representations of possible Ugo1 structures. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.
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fig1: Analysis of Ugo1 topology. (A and B) Mitochondria were left intact (lanes 5 and 10), converted to mitoplasts (lanes 3 and 8; mitoplast supernatant, lanes 4 and 9), or solubilized (lanes 2 and 7) before treatment with (+) or without (−) trypsin (lanes 1 and 6) and were analyzed by SDS-PAGE and immunoblotting with the indicated antisera. The 23-kD protected C-terminal Ugo1 fragment is indicated by a single arrow, and the 17–20-kD protected N-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the secondary antibody with trypsin (HRP-α rabbit). (C) Schematic of Ugo1 structure. Black boxes designate hydrophobic regions predicted to be bona fide TMDs based on data in A, and gray boxes represent additional possible TMDs. Amino acid positions are listed above the boxes. Predicted cytosolic (cyt) and IMS regions are also indicated. (D) Schematic representations of possible Ugo1 structures. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.

Mentions: We examined the topology of Ugo1 by treating isolated mitochondria with the exogenous protease trypsin. Mitochondria were isolated from a strain expressing a functional C-terminal influenza HA epitope-tagged Ugo1 (Wong et al., 2003). Consistent with published observations, in intact mitochondria, we detected a protected C-terminal fragment of Ugo1 of ∼23 kD, which indicates that the C terminus resides in an internal compartment (Fig. 1 A, compare lane 1 with lane 5; Sesaki and Jensen, 2001). To further explore Ugo1 topology, we used a polyclonal antibody to the N-terminal 125 amino acids of Ugo1 and observed that several Ugo1 fragments in the range of 17–20 kD were protected from trypsin digestion in intact mitochondria (Fig. 1 A, compare lane 6 with lane 10). Under these conditions, the outer membrane marker Fzo1 was digested completely, indicating that the cytosolic face of the outer membrane was fully accessible to the protease (Fig. 1 B). In addition, both IMS (cytochrome b2 and the C-terminal soluble domain of Tim23) and matrix (Abf2) marker proteins were protected from proteolysis, confirming that the mitochondrial outer and inner membranes were intact (Fig. 1 B). However, when both inner and outer mitochondrial membranes were disrupted by treatment with detergent, complete proteolysis of Ugo1 was observed, as detected by both polyclonal and anti-HA antibodies and marker proteins in all mitochondrial compartments (Fig. 1, A and B).


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)

Analysis of Ugo1 topology. (A and B) Mitochondria were left intact (lanes 5 and 10), converted to mitoplasts (lanes 3 and 8; mitoplast supernatant, lanes 4 and 9), or solubilized (lanes 2 and 7) before treatment with (+) or without (−) trypsin (lanes 1 and 6) and were analyzed by SDS-PAGE and immunoblotting with the indicated antisera. The 23-kD protected C-terminal Ugo1 fragment is indicated by a single arrow, and the 17–20-kD protected N-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the secondary antibody with trypsin (HRP-α rabbit). (C) Schematic of Ugo1 structure. Black boxes designate hydrophobic regions predicted to be bona fide TMDs based on data in A, and gray boxes represent additional possible TMDs. Amino acid positions are listed above the boxes. Predicted cytosolic (cyt) and IMS regions are also indicated. (D) Schematic representations of possible Ugo1 structures. 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

fig1: Analysis of Ugo1 topology. (A and B) Mitochondria were left intact (lanes 5 and 10), converted to mitoplasts (lanes 3 and 8; mitoplast supernatant, lanes 4 and 9), or solubilized (lanes 2 and 7) before treatment with (+) or without (−) trypsin (lanes 1 and 6) and were analyzed by SDS-PAGE and immunoblotting with the indicated antisera. The 23-kD protected C-terminal Ugo1 fragment is indicated by a single arrow, and the 17–20-kD protected N-terminal fragments are indicated by asterisks. The double arrow indicates a nonspecific interaction of the secondary antibody with trypsin (HRP-α rabbit). (C) Schematic of Ugo1 structure. Black boxes designate hydrophobic regions predicted to be bona fide TMDs based on data in A, and gray boxes represent additional possible TMDs. Amino acid positions are listed above the boxes. Predicted cytosolic (cyt) and IMS regions are also indicated. (D) Schematic representations of possible Ugo1 structures. MP, mitoplast; MP sup, mitoplast supernatant. N, N terminus; C, C terminus.
Mentions: We examined the topology of Ugo1 by treating isolated mitochondria with the exogenous protease trypsin. Mitochondria were isolated from a strain expressing a functional C-terminal influenza HA epitope-tagged Ugo1 (Wong et al., 2003). Consistent with published observations, in intact mitochondria, we detected a protected C-terminal fragment of Ugo1 of ∼23 kD, which indicates that the C terminus resides in an internal compartment (Fig. 1 A, compare lane 1 with lane 5; Sesaki and Jensen, 2001). To further explore Ugo1 topology, we used a polyclonal antibody to the N-terminal 125 amino acids of Ugo1 and observed that several Ugo1 fragments in the range of 17–20 kD were protected from trypsin digestion in intact mitochondria (Fig. 1 A, compare lane 6 with lane 10). Under these conditions, the outer membrane marker Fzo1 was digested completely, indicating that the cytosolic face of the outer membrane was fully accessible to the protease (Fig. 1 B). In addition, both IMS (cytochrome b2 and the C-terminal soluble domain of Tim23) and matrix (Abf2) marker proteins were protected from proteolysis, confirming that the mitochondrial outer and inner membranes were intact (Fig. 1 B). However, when both inner and outer mitochondrial membranes were disrupted by treatment with detergent, complete proteolysis of Ugo1 was observed, as detected by both polyclonal and anti-HA antibodies and marker proteins in all mitochondrial compartments (Fig. 1, A and B).

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