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Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission.

Richter V, Palmer CS, Osellame LD, Singh AP, Elgass K, Stroud DA, Sesaki H, Kvansakul M, Ryan MT - J. Cell Biol. (2014)

Bottom Line: MiD51 mutants unable to bind nucleotides were still able to recruit Drp1.Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci.MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and 2 Australian Research Council Centre of Excellence in Coherent X-Ray Science, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

ABSTRACT
Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.

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Formation of MiD51 foci is dependent on Drp1. (A) MiD51TALEN MEFs expressing GFP-tagged Miro1, MiD51, MiD51NBD, MiD51ΔPEYFP, MiD51R234A, or MiD51R235A (green) were fixed and immunostained for Drp1 (red). Merged images show the absence or presence of foci at mitochondria. Bars, 10 µm. (B) Wild-type (WT) and Drp1−/− MEFs were transfected with MiD51-GFP (green) and fixed and immunostained for Tom20 (red). Bars: (merge) 20 µm; (inset) 5 µm. Dashed boxes represent insets shown in the far right panel. (C) Time-lapse imaging series of COS-7 cells transfected with MiD51-GFP (green) and stained for mitochondria with MitoTracker deep red. Arrowheads indicate the site of a fission event. Bar, 2 µm.
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fig4: Formation of MiD51 foci is dependent on Drp1. (A) MiD51TALEN MEFs expressing GFP-tagged Miro1, MiD51, MiD51NBD, MiD51ΔPEYFP, MiD51R234A, or MiD51R235A (green) were fixed and immunostained for Drp1 (red). Merged images show the absence or presence of foci at mitochondria. Bars, 10 µm. (B) Wild-type (WT) and Drp1−/− MEFs were transfected with MiD51-GFP (green) and fixed and immunostained for Tom20 (red). Bars: (merge) 20 µm; (inset) 5 µm. Dashed boxes represent insets shown in the far right panel. (C) Time-lapse imaging series of COS-7 cells transfected with MiD51-GFP (green) and stained for mitochondria with MitoTracker deep red. Arrowheads indicate the site of a fission event. Bar, 2 µm.

Mentions: MiD51 and MiD49 can be found at foci at mitochondrial constriction sites, consistent with their role in fission (Palmer et al., 2011). Interestingly, MiD51 mutants still able to recruit Drp1 (e.g., MiD51NBD and MiD51R234A) also retained their ability to form foci at mitochondria, yet mutants MiD51ΔPEYFP and MiD51R235A that are deficient in Drp1 recruitment ability did not form foci and were instead diffusely distributed along the outer membrane like that of the control GFP-Miro1 (Fig. 4 A). We expressed MiD51-GFP in Drp1−/− MEFs (Wakabayashi et al., 2009) and found that it also did not assemble into foci and was instead distributed along the extended mitochondrial filaments (Fig. 4 B). We conclude that MiD49/51 assembly into foci is dependent on the presence of Drp1. Moreover, live-cell imaging of MiD51-GFP foci revealed that fission events indeed occurred at these sites and that a population of MiD51 in foci was inherited by each daughter organelle (Fig. 4 C and Videos 1 and 2). The distribution of MiD51 foci between organelles after a successful scission event also resembles that of yeast Drp1, Dnm1 (Bleazard et al., 1999). In yeast, Fis1 is a membrane receptor that helps assemble Mdv1p onto the mitochondrial surface where Mdv1p facilitates Dnm1 assembly into scission complexes (Tieu and Nunnari, 2000; Karren et al., 2005). Thus, MiD51 appears to act in a similar way to Mdv1p, yet it does not require an additional receptor because it is already anchored in the mitochondrial outer membrane. Both MiD51 and Mdv1p have different structural folds and hence the final scission complexes are likely to adopt different topologies. Consistent with this, it was recently proposed that variations in the sequence of the Insert B region of dynamin-related proteins accommodates diversity in adaptor binding (Ford et al., 2011; Bui and Shaw, 2013).


Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission.

Richter V, Palmer CS, Osellame LD, Singh AP, Elgass K, Stroud DA, Sesaki H, Kvansakul M, Ryan MT - J. Cell Biol. (2014)

Formation of MiD51 foci is dependent on Drp1. (A) MiD51TALEN MEFs expressing GFP-tagged Miro1, MiD51, MiD51NBD, MiD51ΔPEYFP, MiD51R234A, or MiD51R235A (green) were fixed and immunostained for Drp1 (red). Merged images show the absence or presence of foci at mitochondria. Bars, 10 µm. (B) Wild-type (WT) and Drp1−/− MEFs were transfected with MiD51-GFP (green) and fixed and immunostained for Tom20 (red). Bars: (merge) 20 µm; (inset) 5 µm. Dashed boxes represent insets shown in the far right panel. (C) Time-lapse imaging series of COS-7 cells transfected with MiD51-GFP (green) and stained for mitochondria with MitoTracker deep red. Arrowheads indicate the site of a fission event. Bar, 2 µm.
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Related In: Results  -  Collection

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fig4: Formation of MiD51 foci is dependent on Drp1. (A) MiD51TALEN MEFs expressing GFP-tagged Miro1, MiD51, MiD51NBD, MiD51ΔPEYFP, MiD51R234A, or MiD51R235A (green) were fixed and immunostained for Drp1 (red). Merged images show the absence or presence of foci at mitochondria. Bars, 10 µm. (B) Wild-type (WT) and Drp1−/− MEFs were transfected with MiD51-GFP (green) and fixed and immunostained for Tom20 (red). Bars: (merge) 20 µm; (inset) 5 µm. Dashed boxes represent insets shown in the far right panel. (C) Time-lapse imaging series of COS-7 cells transfected with MiD51-GFP (green) and stained for mitochondria with MitoTracker deep red. Arrowheads indicate the site of a fission event. Bar, 2 µm.
Mentions: MiD51 and MiD49 can be found at foci at mitochondrial constriction sites, consistent with their role in fission (Palmer et al., 2011). Interestingly, MiD51 mutants still able to recruit Drp1 (e.g., MiD51NBD and MiD51R234A) also retained their ability to form foci at mitochondria, yet mutants MiD51ΔPEYFP and MiD51R235A that are deficient in Drp1 recruitment ability did not form foci and were instead diffusely distributed along the outer membrane like that of the control GFP-Miro1 (Fig. 4 A). We expressed MiD51-GFP in Drp1−/− MEFs (Wakabayashi et al., 2009) and found that it also did not assemble into foci and was instead distributed along the extended mitochondrial filaments (Fig. 4 B). We conclude that MiD49/51 assembly into foci is dependent on the presence of Drp1. Moreover, live-cell imaging of MiD51-GFP foci revealed that fission events indeed occurred at these sites and that a population of MiD51 in foci was inherited by each daughter organelle (Fig. 4 C and Videos 1 and 2). The distribution of MiD51 foci between organelles after a successful scission event also resembles that of yeast Drp1, Dnm1 (Bleazard et al., 1999). In yeast, Fis1 is a membrane receptor that helps assemble Mdv1p onto the mitochondrial surface where Mdv1p facilitates Dnm1 assembly into scission complexes (Tieu and Nunnari, 2000; Karren et al., 2005). Thus, MiD51 appears to act in a similar way to Mdv1p, yet it does not require an additional receptor because it is already anchored in the mitochondrial outer membrane. Both MiD51 and Mdv1p have different structural folds and hence the final scission complexes are likely to adopt different topologies. Consistent with this, it was recently proposed that variations in the sequence of the Insert B region of dynamin-related proteins accommodates diversity in adaptor binding (Ford et al., 2011; Bui and Shaw, 2013).

Bottom Line: MiD51 mutants unable to bind nucleotides were still able to recruit Drp1.Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci.MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and 2 Australian Research Council Centre of Excellence in Coherent X-Ray Science, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

ABSTRACT
Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.

Show MeSH
Related in: MedlinePlus