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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 GFP-Mdv1p localization defects that are suppressed by the E250G substitution. DIC (left), GFP-Mdv1p or GFP-Mdv1E250Gp (middle), and GFP-Mdv1p or GFP-Mdv1E250Gp merged with mt-RFP (right) images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with mitochondrial localization of GFP-Mdv1p or GFP-Mdv1E250Gp at each temperature (n ≥ 300; SDs are indicated). (A) GFP-Mdv1p localization in FIS1 mdv1Δ (top), fis1Δ mdv1Δ (middle), and fis1-3 mdv1Δ cells (bottom). (B) GFP-Mdv1E250Gp localization in fis1-3 mdv1Δ cells. To maximize visibility of GFP-Mdv1p, we used mdv1Δ cells expressing plasmid-borne GFP-Mdv1p from the MET25 promoter without induction. Bar, 5 μm.
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fig3: fis1-3 mutations cause GFP-Mdv1p localization defects that are suppressed by the E250G substitution. DIC (left), GFP-Mdv1p or GFP-Mdv1E250Gp (middle), and GFP-Mdv1p or GFP-Mdv1E250Gp merged with mt-RFP (right) images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with mitochondrial localization of GFP-Mdv1p or GFP-Mdv1E250Gp at each temperature (n ≥ 300; SDs are indicated). (A) GFP-Mdv1p localization in FIS1 mdv1Δ (top), fis1Δ mdv1Δ (middle), and fis1-3 mdv1Δ cells (bottom). (B) GFP-Mdv1E250Gp localization in fis1-3 mdv1Δ cells. To maximize visibility of GFP-Mdv1p, we used mdv1Δ cells expressing plasmid-borne GFP-Mdv1p from the MET25 promoter without induction. Bar, 5 μm.

Mentions: Localization of functional GFP-Mdv1p to mitochondria is also significantly reduced in fis1-3 mdv1Δ cells at both temperatures (Fig. 3 A). In wild-type cells, GFP-Mdv1p localizes in punctate fission complexes (Tieu and Nunnari, 2000; Cerveny et al., 2001) and is sometimes also uniformly distributed on mitochondrial tubules (unpublished data). Both localization patterns were scored as punctate GFP-Mdv1p localization in this study. In the majority of FIS1 mdv1Δ cells, punctate GFP-Mdv1p colocalizes with mitochondria at both temperatures. However, GFP-Mdv1p puncta on mitochondria are only observed in 49% of fis1-3 mdv1Δ cells at 25°C. These puncta are often nearly obscured by cytosolic fluorescence not present in FIS1 control cells expressing equivalent amounts of GFP-Mdv1p (unpublished data). At 37°C, GFP-Mdv1p localizes almost entirely to the cytoplasm in fis1-3 mdv1Δ cells, similar to the GFP-Mdv1p localization pattern in fis1Δ mdv1Δ cells. These studies demonstrate that Dnm1-GFP and GFP-Mdv1p do not assemble into mitochondrial fission complexes in fis1-3 cells at 37°C.


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 GFP-Mdv1p localization defects that are suppressed by the E250G substitution. DIC (left), GFP-Mdv1p or GFP-Mdv1E250Gp (middle), and GFP-Mdv1p or GFP-Mdv1E250Gp merged with mt-RFP (right) images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with mitochondrial localization of GFP-Mdv1p or GFP-Mdv1E250Gp at each temperature (n ≥ 300; SDs are indicated). (A) GFP-Mdv1p localization in FIS1 mdv1Δ (top), fis1Δ mdv1Δ (middle), and fis1-3 mdv1Δ cells (bottom). (B) GFP-Mdv1E250Gp localization in fis1-3 mdv1Δ cells. To maximize visibility of GFP-Mdv1p, we used mdv1Δ cells expressing plasmid-borne GFP-Mdv1p from the MET25 promoter without induction. Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171191&req=5

fig3: fis1-3 mutations cause GFP-Mdv1p localization defects that are suppressed by the E250G substitution. DIC (left), GFP-Mdv1p or GFP-Mdv1E250Gp (middle), and GFP-Mdv1p or GFP-Mdv1E250Gp merged with mt-RFP (right) images of indicated strains grown at 37°C are shown. Histograms indicate the percentage of cells with mitochondrial localization of GFP-Mdv1p or GFP-Mdv1E250Gp at each temperature (n ≥ 300; SDs are indicated). (A) GFP-Mdv1p localization in FIS1 mdv1Δ (top), fis1Δ mdv1Δ (middle), and fis1-3 mdv1Δ cells (bottom). (B) GFP-Mdv1E250Gp localization in fis1-3 mdv1Δ cells. To maximize visibility of GFP-Mdv1p, we used mdv1Δ cells expressing plasmid-borne GFP-Mdv1p from the MET25 promoter without induction. Bar, 5 μm.
Mentions: Localization of functional GFP-Mdv1p to mitochondria is also significantly reduced in fis1-3 mdv1Δ cells at both temperatures (Fig. 3 A). In wild-type cells, GFP-Mdv1p localizes in punctate fission complexes (Tieu and Nunnari, 2000; Cerveny et al., 2001) and is sometimes also uniformly distributed on mitochondrial tubules (unpublished data). Both localization patterns were scored as punctate GFP-Mdv1p localization in this study. In the majority of FIS1 mdv1Δ cells, punctate GFP-Mdv1p colocalizes with mitochondria at both temperatures. However, GFP-Mdv1p puncta on mitochondria are only observed in 49% of fis1-3 mdv1Δ cells at 25°C. These puncta are often nearly obscured by cytosolic fluorescence not present in FIS1 control cells expressing equivalent amounts of GFP-Mdv1p (unpublished data). At 37°C, GFP-Mdv1p localizes almost entirely to the cytoplasm in fis1-3 mdv1Δ cells, similar to the GFP-Mdv1p localization pattern in fis1Δ mdv1Δ cells. These studies demonstrate that Dnm1-GFP and GFP-Mdv1p do not assemble into mitochondrial fission complexes in fis1-3 cells at 37°C.

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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