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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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Mitochondrial fission can occur in the absence of the Fis1p NH2-terminal arm. (A) Mitochondrial morphology in cells expressing Fis115–155p and Mdv1p from the MET25 promoter. Panels contain superimposed DIC and mt-RFP images representative of phenotypes observed at 25°C. The percentage of cells in the population with fission-competent mitochondria is shown below each panel (n ≥ 300; ± indicates SD). Bar, 5 μm. (B) Quantification of GFP-Mdv1p or GFP-Mdv1E250Gp localization with (black bars) or without (gray bars) MET25 induction (30 min). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne under the control of the MET25 promoter (n ≥ 300; SDs are indicated).
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fig9: Mitochondrial fission can occur in the absence of the Fis1p NH2-terminal arm. (A) Mitochondrial morphology in cells expressing Fis115–155p and Mdv1p from the MET25 promoter. Panels contain superimposed DIC and mt-RFP images representative of phenotypes observed at 25°C. The percentage of cells in the population with fission-competent mitochondria is shown below each panel (n ≥ 300; ± indicates SD). Bar, 5 μm. (B) Quantification of GFP-Mdv1p or GFP-Mdv1E250Gp localization with (black bars) or without (gray bars) MET25 induction (30 min). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne under the control of the MET25 promoter (n ≥ 300; SDs are indicated).

Mentions: To further define the functions of the Fis1p NH2-terminal arm and TPR-like domain, we evaluated mitochondrial morphology and fission complex assembly in cells expressing a Fis1 mutant lacking the NH2-terminal arm (Fis115–155p). Fis115–155p alone or Fis115–155p and either Mdv1p or Mdv1E250Gp were expressed under control of the inducible MET25 promoter using a 30-min induction period, and mitochondrial morphology was quantified. Consistent with a previous study (Suzuki et al., 2005), <10% of fis1Δ cells expressing Fis115–155p and endogenous Mdv1p have fission-competent mitochondria (Fig. 9 A). However, when both Fis115–155p and Mdv1p are simultaneously induced from MET25 promoters in fis1Δ mdv1Δ cells, 45% of cells contain fission-competent mitochondria. In control experiments, 58% of fis1Δ mdv1Δ cells expressing wild-type Fis1p and Mdv1p contain fission-competent mitochondria. Interestingly, Mdv1E250Gp and Mdv1p rescue mitochondrial fission defects to the same extent in this experiment (unpublished data).


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

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

Mitochondrial fission can occur in the absence of the Fis1p NH2-terminal arm. (A) Mitochondrial morphology in cells expressing Fis115–155p and Mdv1p from the MET25 promoter. Panels contain superimposed DIC and mt-RFP images representative of phenotypes observed at 25°C. The percentage of cells in the population with fission-competent mitochondria is shown below each panel (n ≥ 300; ± indicates SD). Bar, 5 μm. (B) Quantification of GFP-Mdv1p or GFP-Mdv1E250Gp localization with (black bars) or without (gray bars) MET25 induction (30 min). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne under the control of the MET25 promoter (n ≥ 300; SDs are indicated).
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Related In: Results  -  Collection

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fig9: Mitochondrial fission can occur in the absence of the Fis1p NH2-terminal arm. (A) Mitochondrial morphology in cells expressing Fis115–155p and Mdv1p from the MET25 promoter. Panels contain superimposed DIC and mt-RFP images representative of phenotypes observed at 25°C. The percentage of cells in the population with fission-competent mitochondria is shown below each panel (n ≥ 300; ± indicates SD). Bar, 5 μm. (B) Quantification of GFP-Mdv1p or GFP-Mdv1E250Gp localization with (black bars) or without (gray bars) MET25 induction (30 min). Genes indicated are genomic unless preceded by “p,” which denotes plasmid borne under the control of the MET25 promoter (n ≥ 300; SDs are indicated).
Mentions: To further define the functions of the Fis1p NH2-terminal arm and TPR-like domain, we evaluated mitochondrial morphology and fission complex assembly in cells expressing a Fis1 mutant lacking the NH2-terminal arm (Fis115–155p). Fis115–155p alone or Fis115–155p and either Mdv1p or Mdv1E250Gp were expressed under control of the inducible MET25 promoter using a 30-min induction period, and mitochondrial morphology was quantified. Consistent with a previous study (Suzuki et al., 2005), <10% of fis1Δ cells expressing Fis115–155p and endogenous Mdv1p have fission-competent mitochondria (Fig. 9 A). However, when both Fis115–155p and Mdv1p are simultaneously induced from MET25 promoters in fis1Δ mdv1Δ cells, 45% of cells contain fission-competent mitochondria. In control experiments, 58% of fis1Δ mdv1Δ cells expressing wild-type Fis1p and Mdv1p contain fission-competent mitochondria. Interestingly, Mdv1E250Gp and Mdv1p rescue mitochondrial fission defects to the same extent in this experiment (unpublished data).

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.

Show MeSH
Related in: MedlinePlus