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.2008 Jun;19(6):2402-12.
doi: 10.1091/mbc.e07-12-1287. Epub 2008 Mar 19.

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells

Affiliations

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells

Shilpa Gandre-Babbe et al. Mol Biol Cell.2008 Jun.

Abstract

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.

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Figures

Figure 1.
Figure 1.
Identification of an Mff homologue in aDrosophila siRNA screen. (A and A′) UntransfectedDrosophila S2 cells stained with MitoTracker. (B and B′) Cells transfected withDrosophila Drp1 siRNA. (C and C′) Cells transfected with CG30404 siRNA. A, B, and C are images taken with a 20× lens in the primary screen (bar, 100 μm), and A′, B′, and C′ are images taken with a 100× lens in the secondary screen (bar, 10 μm). (D) Alignment of theDrosophila CG30404 protein with Mff, which is encoded by C2ORF33. Positions of exon boundaries of the human gene, as deduced from alignment with genomic sequences, are shown below the sequence and key features of the proteins are shown above. Alternative translation start sites identified in different cDNAs are marked with asterisks. (E) Schematic drawing of domains and splice variants identified with different cDNAs encoding Mff. The numbering refers to the exons shown in the alignment in D. The National Center for Biotechnology Information shows two additional isoforms, but those are most likely incomplete or aberrant clones, because they are exceedingly short and represented by single ESTs.
Figure 2.
Figure 2.
Fluorescence and biochemical analysis of Mff localization. (A–C) Immunofluorescence of endogenous protein in HeLa cells. (D–F) Overexpression of myc-tagged Mff. The construct used for this experiment encodes isoform 8, which lacks exons 5, 6, and 7 (Figure 1). A and D show Mff or myc antibody staining, B and E show MitoTracker staining, and C and F show merged images with Mff or myc in green and MitoTracker in red. Bar, 10 μm. (G) Western blots of differential centrifugation fractions prepared from HeLa cells. Mff is present in the low speed supernatant (S1) and the medium speed pellet (P2), which contain mitochondria as shown with Tom20 antibody. Tubulin, which was used to track soluble proteins, is also present as a contaminant in the P2 fraction, but very little Mff is present in the S2 fraction, consistent with mitochondrial localization. The Mff antibody detects bands ranging in size from 25 to 39 kDa, most likely corresponding to different splice variants. (H) Protease protection experiment using the mitochondrial (P2) fraction from bovine brain to determine the topology of Mff. Our Mff antibody detects only a single band of 38-kDa in brain extracts, similar in size to the top band in HeLa cells (see G), suggesting that the repertoire of Mff isoforms is more limited in brain than it is in HeLa cells. The P2 fraction was subjected to increasing amounts of trypsin. Proteins that are exposed to the cytosol, like Tom20, are digested at the lowest concentrations, whereas proteins that are protected by membrane, such as the mitochondrial intermembrane space proteins prohibitin and cytochromec are still protected at the highest concentration. Solubilization with detergents was used to verify that trypsin is able to digest prohibitin and cytochromec were it not for protection by membrane (data not shown). Together, these data show that Mff is exposed to the cytosol. (I) Alkaline extraction shows that Mff is anchored in membrane. The P2 fraction from bovine brain extracts was resuspended in carbonate buffer, pH 11.5. Membranes were pelleted by centrifugation at 100,000 ×g. Tom20 serves as marker for the membrane fraction, and cytochromec as marker for the cytosolic fraction. These experiments show that Mff cofractionates with mitochondria, that it is exposed to the cytosol, and that it is a membrane-anchored protein.
Figure 3.
Figure 3.
Effects of Mff siRNA on mitochondrial morphology. (A) HeLa cell transfected with scrambled Mff siRNA oligonucleotides and stained with MitoTracker. These cells show wild-type mitochondrial morphologies. (B) HeLa cell transfected with Mff, (C) with Drp1 siRNA and (D) with Fis1 siRNA oligonucleotides. These transfected cells all show highly connected mitochondria, consistent with defects in mitochondrial fission. Bar, 10 μm. (E) Western blots showing protein levels in siRNA-transfected cells. The blots show reduced levels in HeLa cells transfected with Mff, Drp1 or Fis1 siRNA, in comparison with scrambled (scr.) controlled. The Mff antibody shows two prominent bands (25 and 35 kDa) and several fainter bands that may correspond to different isoforms produced by alternative splicing. Tubulin serves as loading control. The levels of Mff are reduced by 88%, of Drp1 by 93%, and of Fis1 by 82% as determined by densitometry.
Figure 4.
Figure 4.
Effects of Mff siRNA on peroxisomal morphology. (A) HeLa cells transfected with scrambled oligonucleotides showing wild-type mitochondrial morphologies by staining with MitoTracker. (B) The same cells stained with catalase antibody, showing the punctate distribution of peroxisomes. The inset shows an enlargement of the peroxisomal staining. (C and D) HeLa cells transfected with Drp1 siRNA oligonucleotides, showing highly connected mitochondria and elongated peroxisomes. The enlargement shows the peroxisomal defect more clearly. (E and F) Similar patterns were obtained with Mff siRNA oligonucleotides. The boxed areas, enlarged in the bottom right-hand corners of D–F, show close-ups of peroxisomes. Bar, 10 μm.
Figure 5.
Figure 5.
Reversal of mitochondrial fragmentation caused by dominant-negative Mitofusin expression constructs. (A) HeLa cells were transfected with scrambled Mff siRNA oligonucleotides as a control and with a myc-Mfn2(K109T) expression construct. Mitochondria were detected with MitoTracker (red). Cells expressing mutant Mfn2 were detected with myc antibody (green). These cells invariably had fragmented and often clumped mitochondria, most likely due to overexpression of aberrant mitochondrial outer membrane protein. Similar transfections were done with Mff (B), Drp1 (C), and Fis1 (D) siRNA oligonucleotides along with the myc-Mfn2(K109T) expression construct. The cotransfected cells show clumped mitochondria, but these mitochondria often have thin tubular connections, which were never detected when the myc-Mfn2(K109T) construct was transfected alone. The enlargements in the bottom left corners show these connections more clearly. Bar, 10 μm. The percentages of cells with mitochondrial connections are shown in E. The experiments numbered 1 were done with GFP tagged Mfn1(K88T), and the experiments numbered 2 and 3 were done with myc-Mfn2(K109T). For each point, 250–400 cells were counted.
Figure 6.
Figure 6.
Reversal of fission induced by loss of mitochondrial membrane potential. HeLa cells were transfected with scrambled, Mff, Fis1, or Drp1 siRNA oligonucleotides, respectively (A–D), stained with MitoTracker, and then incubated for 60 min with the membrane-depolarizing drug CCCP at 2 μg/ml to induce mitochondrial fission (E–H). Transfection with Drp1 siRNA inhibits almost all of the induced fragmentation. Transfection with Mff siRNA inhibits some, but not all, of the induced fragmentation, whereas transfection with Fis1 siRNA had only a limited effect. Bar, 10 μm. I shows quantification of these effects. N, highly connected tubular networks; T, mixture of tubules as observed in untreated cells; S, short tubules; and R, round fragments. The percentages are means of three independent experiments with 300–400 cells per data point. The error bars show SD for the three experiments. The bars show values obtained after CCCP induction. Although Drp1 siRNA is the only one that strongly inhibits CCCP-induced fission, the effects of Mff and Fis1 siRNA are significant when the percentages of cells with round fragments are compared with those percentages in cells transfected with scrambled oligonucleotides. An unpaired Student'st test shows p < 0.0005 for Mff (*), p < 0.005 for Fis1 (**), and p < 0.0001 for Drp1 (***). The amounts of Mff protein were reduced by 91%, Fis1 by 93%, and Drp1 by 99% (average values as determined by densitometry of Western blots).
Figure 7.
Figure 7.
Inhibition of apoptosis by Mff siRNA. HeLa cells were transfected with scrambled, Mff, Fis1, and Drp1 siRNA oligonucleotides as indicated, and apoptosis was induced by treating the cells with staurosporine or as control with solvent (dimethyl sulfoxide). Cells were fixed and stained at fixed intervals after the addition of staurosporine as indicated. (A) Effects on cytochromec release from mitochondria as detected by staining the cells with cytochromec antibody. Along with Staurosporine, z-VAD-fmk was added to prevent further progression of apoptosis. (B) Effects on the formation of apoptotic nuclei as detected with Hoechst staining. No z-VAD-fmk was added to allow further progression of apoptosis. The percentages are means of three independent experiments, each with 300–400 cells per data point. The error bars show SD for these three experiments. An unpaired Student'st test was used for statistical analysis of data collected after 90 min with staurosporine.
Figure 8.
Figure 8.
Multimers of Mff and endogenous Mff complexes. (A) Coimmunoprecipitations to determine whether Mff can form homomultimers. HEK293 cells were transfected with myc- and GFP-tagged Mff expression constructs, the transfected cells were lysed, and the lysates were incubated with anti-myc antibody coupled to protein A beads and immunoprecipitated. The blot was probed with Mff antibody, showing coimmunpreciptation of GFP-tagged Mff with anti-myc antibody when the GFP and myc-tagged constructs are cotransfected, but not when the GFP-tagged construct is transfected alone. The lanes with total lysates verify that the constructs are expressed. The constructs used for these experiments encode isoform 8, which lacks exons 5, 6, and 7 (Figure 1). (B) Size of native Mff complex determined with Blue Native Gel electrophoresis. Detergent lysates from cells transfected with scrambled, Fis1 siRNA, and Mff siRNA oligonucleotides were subjected to BNGE, blotted, and probed with Drp1, Mff, and Fis1 antibodies.
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