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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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Quantification of mitochondrial outer and inner membrane tethering efficiency. (A) Comparison of the abundance of tethered mitochondria observed in mitochondria isolated from UGO1, ugo1ts, and fzo1-1 strains subjected to S1 in vitro fusion conditions. Wild-type mitochondria were also either treated with the nonhydrolysable GTP derivative PCP or pretreated with trypsin to remove cytosolic portions of outer membrane proteins before S1 in vitro fusion conditions. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 4.5; ugo1-1, 7.9; ugo1-2, 6.3; ugo1-3, 6.3; ugo1-4, 5.3; ugo1-5, 3.4; fzo1-1, 8.4; PCP, 6.3; and trypsinized, 1.98. (B) Comparison of the abundance of tight inner membrane interfaces in outer membrane–fused structure observed in the in vitro fusion mitochondria isolated from UGO1, ugo1-4, ugo1-5, and mgm1-10 strains. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 80; ugo1-4, 100; ugo1-5, 100; and mgm1-10, 51. White bars show wild-type control strains. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane. Data are represented as mean ± SEM.
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fig7: Quantification of mitochondrial outer and inner membrane tethering efficiency. (A) Comparison of the abundance of tethered mitochondria observed in mitochondria isolated from UGO1, ugo1ts, and fzo1-1 strains subjected to S1 in vitro fusion conditions. Wild-type mitochondria were also either treated with the nonhydrolysable GTP derivative PCP or pretreated with trypsin to remove cytosolic portions of outer membrane proteins before S1 in vitro fusion conditions. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 4.5; ugo1-1, 7.9; ugo1-2, 6.3; ugo1-3, 6.3; ugo1-4, 5.3; ugo1-5, 3.4; fzo1-1, 8.4; PCP, 6.3; and trypsinized, 1.98. (B) Comparison of the abundance of tight inner membrane interfaces in outer membrane–fused structure observed in the in vitro fusion mitochondria isolated from UGO1, ugo1-4, ugo1-5, and mgm1-10 strains. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 80; ugo1-4, 100; ugo1-5, 100; and mgm1-10, 51. White bars show wild-type control strains. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane. Data are represented as mean ± SEM.

Mentions: We looked for outer membrane–tethered structures that were functionally relevant for mitochondrial fusion in our yeast in vitro assay under S1 conditions. Under S1 conditions with wild-type mitochondria, EM analysis revealed a population of closely associated mitochondria (22%, n = 350; Fig. 6 A). To discern structures that were engaged from those that were touching indiscriminately, we counted only those with distinct morphological features consistent with membrane-tethered intermediates (4.5%, n = 350; Fig. 6 A). Specifically, in regions of mitochondrial association, we looked for the outer membranes to be reciprocally deformed and evenly spaced apart with an electron-dense region between them (Fig. 6, A [arrows] and B [boxes]). These structures were less abundant under S2 conditions that promote both outer and inner membrane fusion, which is consistent with the interpretation that they are productive intermediates (1.6%, n = 400). To further determine whether these structures are bona fide outer membrane–tethered intermediates, we localized Fzo1 by immuno-EM analysis of wild-type mitochondria under S1 assay conditions. Consistent with its proposed role in outer membrane tethering, we observed that Fzo1 is localized on the outer membrane and is enriched at the interface of adjacent tethered mitochondria (Fig. 6 C, gold particles). In addition, S1 reactions containing fzo1-1 and wild-type mitochondria treated with the nonhydrolyzable GTP analogue GMP-PCP (β,γ-methyleneguanosine 5′-triphosphate) accumulated outer membrane–tethered structures to levels greater than those observed in wild-type S1 reactions, which is consistent with being blocked at a step preceding outer membrane fusion (Fig. 7 A; Meeusen et al., 2004). In contrast, treatment of wild-type mitochondria with the protease trypsin significantly decreased the amount of outer membrane–tethered structures observed (Fig. 7 A). Collectively, these observations indicate that we have identified outer membrane–tethered intermediates in the pathway of fusion and are consistent with the proposed role of Fzo1 in outer membrane tethering.


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)

Quantification of mitochondrial outer and inner membrane tethering efficiency. (A) Comparison of the abundance of tethered mitochondria observed in mitochondria isolated from UGO1, ugo1ts, and fzo1-1 strains subjected to S1 in vitro fusion conditions. Wild-type mitochondria were also either treated with the nonhydrolysable GTP derivative PCP or pretreated with trypsin to remove cytosolic portions of outer membrane proteins before S1 in vitro fusion conditions. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 4.5; ugo1-1, 7.9; ugo1-2, 6.3; ugo1-3, 6.3; ugo1-4, 5.3; ugo1-5, 3.4; fzo1-1, 8.4; PCP, 6.3; and trypsinized, 1.98. (B) Comparison of the abundance of tight inner membrane interfaces in outer membrane–fused structure observed in the in vitro fusion mitochondria isolated from UGO1, ugo1-4, ugo1-5, and mgm1-10 strains. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 80; ugo1-4, 100; ugo1-5, 100; and mgm1-10, 51. White bars show wild-type control strains. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane. Data are represented as mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Quantification of mitochondrial outer and inner membrane tethering efficiency. (A) Comparison of the abundance of tethered mitochondria observed in mitochondria isolated from UGO1, ugo1ts, and fzo1-1 strains subjected to S1 in vitro fusion conditions. Wild-type mitochondria were also either treated with the nonhydrolysable GTP derivative PCP or pretreated with trypsin to remove cytosolic portions of outer membrane proteins before S1 in vitro fusion conditions. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 4.5; ugo1-1, 7.9; ugo1-2, 6.3; ugo1-3, 6.3; ugo1-4, 5.3; ugo1-5, 3.4; fzo1-1, 8.4; PCP, 6.3; and trypsinized, 1.98. (B) Comparison of the abundance of tight inner membrane interfaces in outer membrane–fused structure observed in the in vitro fusion mitochondria isolated from UGO1, ugo1-4, ugo1-5, and mgm1-10 strains. The absolute values of the mean of at least two independent experiments (percentage of total mitochondria, n > 350) are UGO1, 80; ugo1-4, 100; ugo1-5, 100; and mgm1-10, 51. White bars show wild-type control strains. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane. Data are represented as mean ± SEM.
Mentions: We looked for outer membrane–tethered structures that were functionally relevant for mitochondrial fusion in our yeast in vitro assay under S1 conditions. Under S1 conditions with wild-type mitochondria, EM analysis revealed a population of closely associated mitochondria (22%, n = 350; Fig. 6 A). To discern structures that were engaged from those that were touching indiscriminately, we counted only those with distinct morphological features consistent with membrane-tethered intermediates (4.5%, n = 350; Fig. 6 A). Specifically, in regions of mitochondrial association, we looked for the outer membranes to be reciprocally deformed and evenly spaced apart with an electron-dense region between them (Fig. 6, A [arrows] and B [boxes]). These structures were less abundant under S2 conditions that promote both outer and inner membrane fusion, which is consistent with the interpretation that they are productive intermediates (1.6%, n = 400). To further determine whether these structures are bona fide outer membrane–tethered intermediates, we localized Fzo1 by immuno-EM analysis of wild-type mitochondria under S1 assay conditions. Consistent with its proposed role in outer membrane tethering, we observed that Fzo1 is localized on the outer membrane and is enriched at the interface of adjacent tethered mitochondria (Fig. 6 C, gold particles). In addition, S1 reactions containing fzo1-1 and wild-type mitochondria treated with the nonhydrolyzable GTP analogue GMP-PCP (β,γ-methyleneguanosine 5′-triphosphate) accumulated outer membrane–tethered structures to levels greater than those observed in wild-type S1 reactions, which is consistent with being blocked at a step preceding outer membrane fusion (Fig. 7 A; Meeusen et al., 2004). In contrast, treatment of wild-type mitochondria with the protease trypsin significantly decreased the amount of outer membrane–tethered structures observed (Fig. 7 A). Collectively, these observations indicate that we have identified outer membrane–tethered intermediates in the pathway of fusion and are consistent with the proposed role of Fzo1 in outer membrane tethering.

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