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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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Fis1-3p is more sensitive to protease than wild-type Fis1p. Limited proteolysis of the wild-type Fis11–12710Hisp (top) and mutant Fis1-31–12710Hisp (bottom). Purified proteins were digested with 0, 0.1, 0.5, 1, 2, 3, 4, or 5 μg of trypsin in a 10-μl reaction vol (see Materials and methods). Digestion was performed at 37°C for 2 min, and samples were analyzed by Western blot using anti-Fis1p antibody. Bracket marks proteolytic fragments. Asterisk marks species lacking 10His tag.
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fig8: Fis1-3p is more sensitive to protease than wild-type Fis1p. Limited proteolysis of the wild-type Fis11–12710Hisp (top) and mutant Fis1-31–12710Hisp (bottom). Purified proteins were digested with 0, 0.1, 0.5, 1, 2, 3, 4, or 5 μg of trypsin in a 10-μl reaction vol (see Materials and methods). Digestion was performed at 37°C for 2 min, and samples were analyzed by Western blot using anti-Fis1p antibody. Bracket marks proteolytic fragments. Asterisk marks species lacking 10His tag.

Mentions: Western blot analysis shows that wild-type Fis11–12710Hisp is extremely resistant to trypsin digestion, with uncut protein persisting at high protease concentrations (Fig. 8, top panel, top band). Digestion with increasing trypsin concentration converts this full-length protein to a stable, lower molecular weight band (Fig. 8, top panel, bottom band). Mass spectrometry of digested samples reveals a mixture of several proteolytic fragments generated predominantly by cleavage at the NH2 and COOH termini (unpublished data). NH2-terminal cleavage sites occur in the first five residues, consistent with nuclear magnetic resonance studies showing that the NH2-terminal five residues of yeast Fis1p are unstructured (Suzuki et al., 2005). COOH-terminal cleavage sites are located throughout the 10His tag used for purification. Importantly, fragments generated by cleavage at sites internal to the protein are rarely observed. Thus, the TPR-like domain and NH2-terminal helix form a highly stable fold, with the most labile portion at the extreme NH2 terminus.


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

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

Fis1-3p is more sensitive to protease than wild-type Fis1p. Limited proteolysis of the wild-type Fis11–12710Hisp (top) and mutant Fis1-31–12710Hisp (bottom). Purified proteins were digested with 0, 0.1, 0.5, 1, 2, 3, 4, or 5 μg of trypsin in a 10-μl reaction vol (see Materials and methods). Digestion was performed at 37°C for 2 min, and samples were analyzed by Western blot using anti-Fis1p antibody. Bracket marks proteolytic fragments. Asterisk marks species lacking 10His tag.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Fis1-3p is more sensitive to protease than wild-type Fis1p. Limited proteolysis of the wild-type Fis11–12710Hisp (top) and mutant Fis1-31–12710Hisp (bottom). Purified proteins were digested with 0, 0.1, 0.5, 1, 2, 3, 4, or 5 μg of trypsin in a 10-μl reaction vol (see Materials and methods). Digestion was performed at 37°C for 2 min, and samples were analyzed by Western blot using anti-Fis1p antibody. Bracket marks proteolytic fragments. Asterisk marks species lacking 10His tag.
Mentions: Western blot analysis shows that wild-type Fis11–12710Hisp is extremely resistant to trypsin digestion, with uncut protein persisting at high protease concentrations (Fig. 8, top panel, top band). Digestion with increasing trypsin concentration converts this full-length protein to a stable, lower molecular weight band (Fig. 8, top panel, bottom band). Mass spectrometry of digested samples reveals a mixture of several proteolytic fragments generated predominantly by cleavage at the NH2 and COOH termini (unpublished data). NH2-terminal cleavage sites occur in the first five residues, consistent with nuclear magnetic resonance studies showing that the NH2-terminal five residues of yeast Fis1p are unstructured (Suzuki et al., 2005). COOH-terminal cleavage sites are located throughout the 10His tag used for purification. Importantly, fragments generated by cleavage at sites internal to the protein are rarely observed. Thus, the TPR-like domain and NH2-terminal helix form a highly stable fold, with the most labile portion at the extreme NH2 terminus.

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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