Limits...
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.

Show MeSH

Related in: MedlinePlus

The Mdv1E250Gp suppressor specifically affects interactions with Fis1-3p. Yeast two-hybrid assays show interactions between Mdv1 and Fis1 proteins (A), between Mdv1 proteins and Dnm1p (B), and between Mdv1 proteins (C). pBD and pAD plasmids carrying the indicated genes were cotransformed into the Y187 yeast two-hybrid reporter strain. Strains were grown on S-dextrose selective plates at 25°C, and a filter lift β-galactosidase assay was performed at either 25 or 37°C (A) or at 25°C (B and C). Blue indicates positive interaction. Mdv1E250Gp also interacts with Fis11–127p in this assay (unpublished data).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171191&req=5

fig6: The Mdv1E250Gp suppressor specifically affects interactions with Fis1-3p. Yeast two-hybrid assays show interactions between Mdv1 and Fis1 proteins (A), between Mdv1 proteins and Dnm1p (B), and between Mdv1 proteins (C). pBD and pAD plasmids carrying the indicated genes were cotransformed into the Y187 yeast two-hybrid reporter strain. Strains were grown on S-dextrose selective plates at 25°C, and a filter lift β-galactosidase assay was performed at either 25 or 37°C (A) or at 25°C (B and C). Blue indicates positive interaction. Mdv1E250Gp also interacts with Fis11–127p in this assay (unpublished data).

Mentions: As published previously (Tieu et al., 2002; Cerveny and Jensen, 2003), Mdv1p fused to the Gal4 activating domain (AD-Mdv1p) interacts with the Fis1p cytoplasmic domain fused to the Gal4 DNA-binding domain (BD-Fis11–127p) to stimulate transcription of GAL4 reporter genes (Fig. 6 A). Although AD-Mdv1p fails to interact with the mutant BD–Fis1-31–127p, the suppressor AD-Mdv1E250Gp interacts robustly with BD–Fis1-31–127p. Introducing the E250G mutation does not affect interaction of Mdv1p with Dnm1p (Fig. 6 B) or with itself (Fig. 6 C) in this assay. In control experiments, constructs paired with empty vectors did not activate transcription. These results are consistent with the idea that the E250G mutation primarily affects heterotypic interactions of Mdv1p with Fis1p, rather than with Dnm1p or with itself. In combination with our coIP experiments, these studies suggest that suppression of Fis1-3p by Mdv1E250Gp is primarily the result of increased interaction between Mdv1 and Fis1 proteins.


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

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

The Mdv1E250Gp suppressor specifically affects interactions with Fis1-3p. Yeast two-hybrid assays show interactions between Mdv1 and Fis1 proteins (A), between Mdv1 proteins and Dnm1p (B), and between Mdv1 proteins (C). pBD and pAD plasmids carrying the indicated genes were cotransformed into the Y187 yeast two-hybrid reporter strain. Strains were grown on S-dextrose selective plates at 25°C, and a filter lift β-galactosidase assay was performed at either 25 or 37°C (A) or at 25°C (B and C). Blue indicates positive interaction. Mdv1E250Gp also interacts with Fis11–127p in this assay (unpublished data).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The Mdv1E250Gp suppressor specifically affects interactions with Fis1-3p. Yeast two-hybrid assays show interactions between Mdv1 and Fis1 proteins (A), between Mdv1 proteins and Dnm1p (B), and between Mdv1 proteins (C). pBD and pAD plasmids carrying the indicated genes were cotransformed into the Y187 yeast two-hybrid reporter strain. Strains were grown on S-dextrose selective plates at 25°C, and a filter lift β-galactosidase assay was performed at either 25 or 37°C (A) or at 25°C (B and C). Blue indicates positive interaction. Mdv1E250Gp also interacts with Fis11–127p in this assay (unpublished data).
Mentions: As published previously (Tieu et al., 2002; Cerveny and Jensen, 2003), Mdv1p fused to the Gal4 activating domain (AD-Mdv1p) interacts with the Fis1p cytoplasmic domain fused to the Gal4 DNA-binding domain (BD-Fis11–127p) to stimulate transcription of GAL4 reporter genes (Fig. 6 A). Although AD-Mdv1p fails to interact with the mutant BD–Fis1-31–127p, the suppressor AD-Mdv1E250Gp interacts robustly with BD–Fis1-31–127p. Introducing the E250G mutation does not affect interaction of Mdv1p with Dnm1p (Fig. 6 B) or with itself (Fig. 6 C) in this assay. In control experiments, constructs paired with empty vectors did not activate transcription. These results are consistent with the idea that the E250G mutation primarily affects heterotypic interactions of Mdv1p with Fis1p, rather than with Dnm1p or with itself. In combination with our coIP experiments, these studies suggest that suppression of Fis1-3p by Mdv1E250Gp is primarily the result of increased interaction between Mdv1 and Fis1 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