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The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1.

Liu R, Chan DC - Mol. Biol. Cell (2015)

Bottom Line: The Drp1 receptor Mff is a major regulator of mitochondrial fission, and its overexpression results in increased fission.In contrast, the alternative Drp1 receptors MiD51 and MiD49 can recruit Drp1 dimers.Therefore Drp1 recruitment by Mff versus MiD51 and MiD49 may result in different outcomes because they recruit different subpopulations of Drp1 from the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.

No MeSH data available.


Related in: MedlinePlus

Identification of Drp1 mutants that fail to bind Mff. (A) Screen of Drp1 mutants by the yeast two-hybrid assay. Full-length Drp1 and Drp1 mutants 1–42 (see Supplemental Table S1) were expressed as AD fusion proteins and tested against Mff104 (Mff fragment 1–104), Mff isoform 4 with its coiled-coil domain deleted (Mff4ΔCC), full-length Drp1, and MiD51 expressed as BD fusions. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Brackets indicate Drp1 mutants that did not interact with Mff, black asterisks denote Drp1 mutants that did not interact with Drp1 (indicating loss of oligomerization ability), and red asterisks denote the subset of Mff-binding–deficient mutants that were selected for further characterization. (B) Secondary screen for Drp1 mutants. The indicated Drp1 mutants were screened against full-length Drp1, Mff104, Mff4ΔCC, and Mff146 (Mff fragment 1–146). The Drp1 mutants were constructed in full-length Drp1 (FL) and Drp1ΔIB. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Mutants highlighted in red from the Drp1ΔIB assay were selected for further characterization. (C) Location of mutations 10, 16, and 17 indicated on the structure of Drp1 (Protein Data Bank file 4BEJ). Loops implicated in higher-order assembly of Drp1 are marked.
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Figure 3: Identification of Drp1 mutants that fail to bind Mff. (A) Screen of Drp1 mutants by the yeast two-hybrid assay. Full-length Drp1 and Drp1 mutants 1–42 (see Supplemental Table S1) were expressed as AD fusion proteins and tested against Mff104 (Mff fragment 1–104), Mff isoform 4 with its coiled-coil domain deleted (Mff4ΔCC), full-length Drp1, and MiD51 expressed as BD fusions. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Brackets indicate Drp1 mutants that did not interact with Mff, black asterisks denote Drp1 mutants that did not interact with Drp1 (indicating loss of oligomerization ability), and red asterisks denote the subset of Mff-binding–deficient mutants that were selected for further characterization. (B) Secondary screen for Drp1 mutants. The indicated Drp1 mutants were screened against full-length Drp1, Mff104, Mff4ΔCC, and Mff146 (Mff fragment 1–146). The Drp1 mutants were constructed in full-length Drp1 (FL) and Drp1ΔIB. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Mutants highlighted in red from the Drp1ΔIB assay were selected for further characterization. (C) Location of mutations 10, 16, and 17 indicated on the structure of Drp1 (Protein Data Bank file 4BEJ). Loops implicated in higher-order assembly of Drp1 are marked.

Mentions: We assayed 42 Drp1 mutants (Figure 3A and Supplemental Table S1) for binding to the Mff, Drp1, and MiD51 baits. Several interesting patterns emerged from the screen. All mutants that affected Drp1 oligomerization, as interpreted by loss of interaction with the Drp1 bait, also abolished binding to Mff (Figure 3A, mutants 8, 9, 15, 18, and 20). In contrast, some of these mutants retained MiD51 binding (mutants 9 and 15). Loss of Drp1 oligomerization therefore correlates tightly with loss of Mff binding but not MiD51 binding. In addition, we recovered several Drp1 mutants that failed to bind Mff but retained binding to both Drp1 and MiD51. We chose six for further study (mutants 10, 13, 16, 17, 22, and 28) and introduced them into a Drp1ΔIB background to test whether removing the insert B domain could rescue the loss of binding (Figure 3B). In three of six mutants, we found recovery of interaction with Mff, consistent with the notion that the insert B domain is inhibitory to the interaction. This secondary screen left us with three mutants that we interpret as bona fide Mff-binding mutants for further characterization (Figure 3C). We tested the three mutants in our in vitro interaction assays to validate the results of the yeast two-hybrid assay. Whereas wild-type Drp1ΔIB binds robustly to Mff(1-61), all three mutants (in the context of Drp1ΔIB) failed to coprecipitate with Mff(1-61) by GST pull down (Figure 4A). Furthermore, by SEC analysis, these mutants showed loss of complex formation with Mff(1-61) (Figure 4B). Taken together, the data suggest that these mutants are indeed defective in Mff binding.


The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1.

Liu R, Chan DC - Mol. Biol. Cell (2015)

Identification of Drp1 mutants that fail to bind Mff. (A) Screen of Drp1 mutants by the yeast two-hybrid assay. Full-length Drp1 and Drp1 mutants 1–42 (see Supplemental Table S1) were expressed as AD fusion proteins and tested against Mff104 (Mff fragment 1–104), Mff isoform 4 with its coiled-coil domain deleted (Mff4ΔCC), full-length Drp1, and MiD51 expressed as BD fusions. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Brackets indicate Drp1 mutants that did not interact with Mff, black asterisks denote Drp1 mutants that did not interact with Drp1 (indicating loss of oligomerization ability), and red asterisks denote the subset of Mff-binding–deficient mutants that were selected for further characterization. (B) Secondary screen for Drp1 mutants. The indicated Drp1 mutants were screened against full-length Drp1, Mff104, Mff4ΔCC, and Mff146 (Mff fragment 1–146). The Drp1 mutants were constructed in full-length Drp1 (FL) and Drp1ΔIB. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Mutants highlighted in red from the Drp1ΔIB assay were selected for further characterization. (C) Location of mutations 10, 16, and 17 indicated on the structure of Drp1 (Protein Data Bank file 4BEJ). Loops implicated in higher-order assembly of Drp1 are marked.
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Figure 3: Identification of Drp1 mutants that fail to bind Mff. (A) Screen of Drp1 mutants by the yeast two-hybrid assay. Full-length Drp1 and Drp1 mutants 1–42 (see Supplemental Table S1) were expressed as AD fusion proteins and tested against Mff104 (Mff fragment 1–104), Mff isoform 4 with its coiled-coil domain deleted (Mff4ΔCC), full-length Drp1, and MiD51 expressed as BD fusions. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Brackets indicate Drp1 mutants that did not interact with Mff, black asterisks denote Drp1 mutants that did not interact with Drp1 (indicating loss of oligomerization ability), and red asterisks denote the subset of Mff-binding–deficient mutants that were selected for further characterization. (B) Secondary screen for Drp1 mutants. The indicated Drp1 mutants were screened against full-length Drp1, Mff104, Mff4ΔCC, and Mff146 (Mff fragment 1–146). The Drp1 mutants were constructed in full-length Drp1 (FL) and Drp1ΔIB. The adenine-deficient plates are shown. The associated diploid selection plates are shown in Supplemental Figure S2. Mutants highlighted in red from the Drp1ΔIB assay were selected for further characterization. (C) Location of mutations 10, 16, and 17 indicated on the structure of Drp1 (Protein Data Bank file 4BEJ). Loops implicated in higher-order assembly of Drp1 are marked.
Mentions: We assayed 42 Drp1 mutants (Figure 3A and Supplemental Table S1) for binding to the Mff, Drp1, and MiD51 baits. Several interesting patterns emerged from the screen. All mutants that affected Drp1 oligomerization, as interpreted by loss of interaction with the Drp1 bait, also abolished binding to Mff (Figure 3A, mutants 8, 9, 15, 18, and 20). In contrast, some of these mutants retained MiD51 binding (mutants 9 and 15). Loss of Drp1 oligomerization therefore correlates tightly with loss of Mff binding but not MiD51 binding. In addition, we recovered several Drp1 mutants that failed to bind Mff but retained binding to both Drp1 and MiD51. We chose six for further study (mutants 10, 13, 16, 17, 22, and 28) and introduced them into a Drp1ΔIB background to test whether removing the insert B domain could rescue the loss of binding (Figure 3B). In three of six mutants, we found recovery of interaction with Mff, consistent with the notion that the insert B domain is inhibitory to the interaction. This secondary screen left us with three mutants that we interpret as bona fide Mff-binding mutants for further characterization (Figure 3C). We tested the three mutants in our in vitro interaction assays to validate the results of the yeast two-hybrid assay. Whereas wild-type Drp1ΔIB binds robustly to Mff(1-61), all three mutants (in the context of Drp1ΔIB) failed to coprecipitate with Mff(1-61) by GST pull down (Figure 4A). Furthermore, by SEC analysis, these mutants showed loss of complex formation with Mff(1-61) (Figure 4B). Taken together, the data suggest that these mutants are indeed defective in Mff binding.

Bottom Line: The Drp1 receptor Mff is a major regulator of mitochondrial fission, and its overexpression results in increased fission.In contrast, the alternative Drp1 receptors MiD51 and MiD49 can recruit Drp1 dimers.Therefore Drp1 recruitment by Mff versus MiD51 and MiD49 may result in different outcomes because they recruit different subpopulations of Drp1 from the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.

No MeSH data available.


Related in: MedlinePlus