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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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Fis1p and Mdv1p interact in the absence of other yeast proteins. pBT and pTRG plasmids carrying the indicated genes were cotransformed into an E. coli two-hybrid reporter strain. Cells were grown at 30°C on M9 medium lacking histidine in the presence or absence of 5 mM 3AT. Growth on M9 − His + 5 mM 3AT indicates a positive interaction.
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fig7: Fis1p and Mdv1p interact in the absence of other yeast proteins. pBT and pTRG plasmids carrying the indicated genes were cotransformed into an E. coli two-hybrid reporter strain. Cells were grown at 30°C on M9 medium lacking histidine in the presence or absence of 5 mM 3AT. Growth on M9 − His + 5 mM 3AT indicates a positive interaction.

Mentions: A mechanistic understanding of fis1-3 suppression by Mdv1E250Gp requires precise knowledge of how the wild-type proteins interact. To date, all experiments evaluating Fis1p–Mdv1p interactions have been performed under conditions where other yeast proteins are present (Tieu et al., 2002; Cerveny and Jensen, 2003). Therefore, these experiments do not determine whether Fis1p–Mdv1p interactions are direct or bridged by other yeast proteins. To address this issue, we performed a two-hybrid assay in bacterial cells (Fig. 7). The fis1 cytoplasmic domain (amino acids 1–127) and full-length MDV1 were cloned into bait plasmids (pBTs) and target plasmids (pTRGs), respectively, and expressed in bacteria. A positive interaction in this assay stimulates transcription of bacterial HIS3. This expression allows cells to grow in histidine-limiting conditions in the presence of 3AT (3-amino-1,2,4-triazole), a competitive inhibitor of the HIS3 gene product. In serial dilution experiments, cells cotransformed with pBT-fis11–127(encoding amino acids 1–127) and pTRG-MDV1 grow significantly better on medium lacking histidine and containing 3AT than cells containing either construct alone. This experiment establishes for the first time that the Fis1p–Mdv1p interaction requires no additional yeast proteins.


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

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

Fis1p and Mdv1p interact in the absence of other yeast proteins. pBT and pTRG plasmids carrying the indicated genes were cotransformed into an E. coli two-hybrid reporter strain. Cells were grown at 30°C on M9 medium lacking histidine in the presence or absence of 5 mM 3AT. Growth on M9 − His + 5 mM 3AT indicates a positive interaction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171191&req=5

fig7: Fis1p and Mdv1p interact in the absence of other yeast proteins. pBT and pTRG plasmids carrying the indicated genes were cotransformed into an E. coli two-hybrid reporter strain. Cells were grown at 30°C on M9 medium lacking histidine in the presence or absence of 5 mM 3AT. Growth on M9 − His + 5 mM 3AT indicates a positive interaction.
Mentions: A mechanistic understanding of fis1-3 suppression by Mdv1E250Gp requires precise knowledge of how the wild-type proteins interact. To date, all experiments evaluating Fis1p–Mdv1p interactions have been performed under conditions where other yeast proteins are present (Tieu et al., 2002; Cerveny and Jensen, 2003). Therefore, these experiments do not determine whether Fis1p–Mdv1p interactions are direct or bridged by other yeast proteins. To address this issue, we performed a two-hybrid assay in bacterial cells (Fig. 7). The fis1 cytoplasmic domain (amino acids 1–127) and full-length MDV1 were cloned into bait plasmids (pBTs) and target plasmids (pTRGs), respectively, and expressed in bacteria. A positive interaction in this assay stimulates transcription of bacterial HIS3. This expression allows cells to grow in histidine-limiting conditions in the presence of 3AT (3-amino-1,2,4-triazole), a competitive inhibitor of the HIS3 gene product. In serial dilution experiments, cells cotransformed with pBT-fis11–127(encoding amino acids 1–127) and pTRG-MDV1 grow significantly better on medium lacking histidine and containing 3AT than cells containing either construct alone. This experiment establishes for the first time that the Fis1p–Mdv1p interaction requires no additional yeast proteins.

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