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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 physical interactions are disrupted by fis1-3 mutations and rescued by Mdv1E250Gp. CoIP experiments demonstrating interactions between Fis1 proteins and Dnm1p or Mdv1 proteins. Fis1 proteins were immunoprecipitated using anti-Myc agarose-conjugated beads. CoIP of Dnm1p or Mdv1 proteins was detected with either native Dnm1p or Mdv1p antibodies (top). Western blots show total loaded (L) and eluted (E) proteins. Immunoprecipitation of 9Myc-Fis1p or 9Myc–Fis1-3p was detected with anti–c-Myc antibodies (middle). The mitochondrial protein porin is provided as a loading control (bottom). (A) Fis1 and Dnm1 proteins coimmunoprecipitated from DSP–cross-linked yeast spheroplasts. (B) Fis1 and Mdv1 proteins coimmunoprecipitated from whole cell lysates in the absence of cross-linking. Asterisk shows that wild-type Mdv1p reproducibly coimmunoprecipitated with wild-type Fis1p at lower levels than Mdv1E250Gp. Arrows denote full-length proteins.
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fig5: Fis1p physical interactions are disrupted by fis1-3 mutations and rescued by Mdv1E250Gp. CoIP experiments demonstrating interactions between Fis1 proteins and Dnm1p or Mdv1 proteins. Fis1 proteins were immunoprecipitated using anti-Myc agarose-conjugated beads. CoIP of Dnm1p or Mdv1 proteins was detected with either native Dnm1p or Mdv1p antibodies (top). Western blots show total loaded (L) and eluted (E) proteins. Immunoprecipitation of 9Myc-Fis1p or 9Myc–Fis1-3p was detected with anti–c-Myc antibodies (middle). The mitochondrial protein porin is provided as a loading control (bottom). (A) Fis1 and Dnm1 proteins coimmunoprecipitated from DSP–cross-linked yeast spheroplasts. (B) Fis1 and Mdv1 proteins coimmunoprecipitated from whole cell lysates in the absence of cross-linking. Asterisk shows that wild-type Mdv1p reproducibly coimmunoprecipitated with wild-type Fis1p at lower levels than Mdv1E250Gp. Arrows denote full-length proteins.

Mentions: 9Myc-Fis1p and 9Myc–Fis1-3p are efficiently immunoprecipitated from DSP-treated cells (Fig. 5 A, anti-Myc, lanes 2, 4, and 6), and a fraction of cellular Dnm1p reproducibly coimmunoprecipitates with 9Myc-Fis1p (Fig. 5 A, anti-Dnm1p, lane 2). Neither Fis1p nor Dnm1p immunoprecipitates when Fis1p lacks the 9Myc tag (Fig. 5 A, anti-Dnm1p and anti-Myc, lane 8), indicating that the precipitation is antibody dependent. Significantly, Dnm1p does not coimmunoprecipitate with 9Myc–Fis1-3p when wild-type Mdv1p is expressed (Fig. 5 A, anti-Dnm1p, lane 4), confirming that the Fis1-3p mutations disrupt Dnm1p–Fis1p complex formation. Expression of Mdv1E250Gp rather than Mdv1p increases the amount of Dnm1p recovered with 9Myc–Fis1-3p (Fig. 5 A, anti-Dnm1p, lane 6), although not to wild-type levels. Together, the in vivo localization and coIP experiments demonstrate that Mdv1E250Gp partially restores Dnm1p association with Fis1-3p in mitochondrial fission complexes.


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

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

Fis1p physical interactions are disrupted by fis1-3 mutations and rescued by Mdv1E250Gp. CoIP experiments demonstrating interactions between Fis1 proteins and Dnm1p or Mdv1 proteins. Fis1 proteins were immunoprecipitated using anti-Myc agarose-conjugated beads. CoIP of Dnm1p or Mdv1 proteins was detected with either native Dnm1p or Mdv1p antibodies (top). Western blots show total loaded (L) and eluted (E) proteins. Immunoprecipitation of 9Myc-Fis1p or 9Myc–Fis1-3p was detected with anti–c-Myc antibodies (middle). The mitochondrial protein porin is provided as a loading control (bottom). (A) Fis1 and Dnm1 proteins coimmunoprecipitated from DSP–cross-linked yeast spheroplasts. (B) Fis1 and Mdv1 proteins coimmunoprecipitated from whole cell lysates in the absence of cross-linking. Asterisk shows that wild-type Mdv1p reproducibly coimmunoprecipitated with wild-type Fis1p at lower levels than Mdv1E250Gp. Arrows denote full-length proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171191&req=5

fig5: Fis1p physical interactions are disrupted by fis1-3 mutations and rescued by Mdv1E250Gp. CoIP experiments demonstrating interactions between Fis1 proteins and Dnm1p or Mdv1 proteins. Fis1 proteins were immunoprecipitated using anti-Myc agarose-conjugated beads. CoIP of Dnm1p or Mdv1 proteins was detected with either native Dnm1p or Mdv1p antibodies (top). Western blots show total loaded (L) and eluted (E) proteins. Immunoprecipitation of 9Myc-Fis1p or 9Myc–Fis1-3p was detected with anti–c-Myc antibodies (middle). The mitochondrial protein porin is provided as a loading control (bottom). (A) Fis1 and Dnm1 proteins coimmunoprecipitated from DSP–cross-linked yeast spheroplasts. (B) Fis1 and Mdv1 proteins coimmunoprecipitated from whole cell lysates in the absence of cross-linking. Asterisk shows that wild-type Mdv1p reproducibly coimmunoprecipitated with wild-type Fis1p at lower levels than Mdv1E250Gp. Arrows denote full-length proteins.
Mentions: 9Myc-Fis1p and 9Myc–Fis1-3p are efficiently immunoprecipitated from DSP-treated cells (Fig. 5 A, anti-Myc, lanes 2, 4, and 6), and a fraction of cellular Dnm1p reproducibly coimmunoprecipitates with 9Myc-Fis1p (Fig. 5 A, anti-Dnm1p, lane 2). Neither Fis1p nor Dnm1p immunoprecipitates when Fis1p lacks the 9Myc tag (Fig. 5 A, anti-Dnm1p and anti-Myc, lane 8), indicating that the precipitation is antibody dependent. Significantly, Dnm1p does not coimmunoprecipitate with 9Myc–Fis1-3p when wild-type Mdv1p is expressed (Fig. 5 A, anti-Dnm1p, lane 4), confirming that the Fis1-3p mutations disrupt Dnm1p–Fis1p complex formation. Expression of Mdv1E250Gp rather than Mdv1p increases the amount of Dnm1p recovered with 9Myc–Fis1-3p (Fig. 5 A, anti-Dnm1p, lane 6), although not to wild-type levels. Together, the in vivo localization and coIP experiments demonstrate that Mdv1E250Gp partially restores Dnm1p association with Fis1-3p in mitochondrial fission complexes.

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