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The role of Fis1p-Mdv1p interactions in mitochondrial fission complex assembly.

Karren MA, Coonrod EM, Anderson TK, Shaw JM - J. Cell Biol. (2005)

Bottom Line: Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil.We also define separable functions for the Fis1p NH(2)-terminal arm and TPR-like fold.These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

ABSTRACT
Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)-like fold and a short NH(2)-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p-Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH(2)-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

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fis1-3 mutations cause Dnm1-GFP localization defects that are suppressed by mdv1E250G. DIC (left), Dnm1-GFP (middle), and Dnm1-GFP merged with mt-RFP (right) representative images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with punctate mitochondrial Dnm1-GFP localization at each temperature (n ≥ 300; SDs are indicated). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne. (A) Dnm1-GFP localization in FIS1 (top), fis1Δ (middle), and fis1-3 cells (bottom). (B) Dnm1-GFP localization in fis1-3 mdv1Δ cells expressing either pRS415MET25-MDV1 (top) or pRS415MET25-mdv1E250G (bottom). Bars, 5 μm.
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fig2: fis1-3 mutations cause Dnm1-GFP localization defects that are suppressed by mdv1E250G. DIC (left), Dnm1-GFP (middle), and Dnm1-GFP merged with mt-RFP (right) representative images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with punctate mitochondrial Dnm1-GFP localization at each temperature (n ≥ 300; SDs are indicated). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne. (A) Dnm1-GFP localization in FIS1 (top), fis1Δ (middle), and fis1-3 cells (bottom). (B) Dnm1-GFP localization in fis1-3 mdv1Δ cells expressing either pRS415MET25-MDV1 (top) or pRS415MET25-mdv1E250G (bottom). Bars, 5 μm.

Mentions: In wild-type cells, Dnm1-GFP in fission complexes appears as punctate structures distributed evenly along mitochondrial tubules (Fig. 2 A). At 25°C, ∼95% of wild-type and fis1-3 cells contain Dnm1-GFP puncta that colocalize with mitochondrial tubules. This localization pattern is maintained in FIS1 wild-type cells shifted to 37°C. Conversely, all visible Dnm1-GFP localizes to the cytoplasm in fis1-3 cells shifted to 37°C, either in rapidly moving dots or in several randomly localized structures, similar to those in fis1Δ cells.


The role of Fis1p-Mdv1p interactions in mitochondrial fission complex assembly.

Karren MA, Coonrod EM, Anderson TK, Shaw JM - J. Cell Biol. (2005)

fis1-3 mutations cause Dnm1-GFP localization defects that are suppressed by mdv1E250G. DIC (left), Dnm1-GFP (middle), and Dnm1-GFP merged with mt-RFP (right) representative images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with punctate mitochondrial Dnm1-GFP localization at each temperature (n ≥ 300; SDs are indicated). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne. (A) Dnm1-GFP localization in FIS1 (top), fis1Δ (middle), and fis1-3 cells (bottom). (B) Dnm1-GFP localization in fis1-3 mdv1Δ cells expressing either pRS415MET25-MDV1 (top) or pRS415MET25-mdv1E250G (bottom). Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171191&req=5

fig2: fis1-3 mutations cause Dnm1-GFP localization defects that are suppressed by mdv1E250G. DIC (left), Dnm1-GFP (middle), and Dnm1-GFP merged with mt-RFP (right) representative images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with punctate mitochondrial Dnm1-GFP localization at each temperature (n ≥ 300; SDs are indicated). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne. (A) Dnm1-GFP localization in FIS1 (top), fis1Δ (middle), and fis1-3 cells (bottom). (B) Dnm1-GFP localization in fis1-3 mdv1Δ cells expressing either pRS415MET25-MDV1 (top) or pRS415MET25-mdv1E250G (bottom). Bars, 5 μm.
Mentions: In wild-type cells, Dnm1-GFP in fission complexes appears as punctate structures distributed evenly along mitochondrial tubules (Fig. 2 A). At 25°C, ∼95% of wild-type and fis1-3 cells contain Dnm1-GFP puncta that colocalize with mitochondrial tubules. This localization pattern is maintained in FIS1 wild-type cells shifted to 37°C. Conversely, all visible Dnm1-GFP localizes to the cytoplasm in fis1-3 cells shifted to 37°C, either in rapidly moving dots or in several randomly localized structures, similar to those in fis1Δ cells.

Bottom Line: Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil.We also define separable functions for the Fis1p NH(2)-terminal arm and TPR-like fold.These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

ABSTRACT
Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)-like fold and a short NH(2)-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p-Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH(2)-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

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