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Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo

Manuel Rojo{ddagger}, Frédéric Legros, Danielle Chateau* and Anne Lombès

INSERM U 523 — Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, 47 boulevard de l'Hôpital, 75651 Paris Cedex 13, France
* Present address: INSERM U 505, Centre de Recherches Biomédicales des Cordeliers, 75006 Paris, France



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  		Fig. 1. Mammalian mitofusins are ubiquitously expressed. (A,B) RT-PCR of mitofusins (Mfn1, Mfn2) and of mitochondrial transcription factor A (Tfam) from human (A) and mouse (B) RNA. Shown are equal aliquots of the amplification reactions after 25, 30 and 35 cycles. Both mitofusins are ubiquitously expressed at similar levels. Tissues: B, brain; F, fibroblasts; H, heart; K, kidney; L, liver; M, muscle; T, Testis. (C) Dendogram of the Fzo-family. Mammalian homologs are called mitofusins (Mfn). Species: Ce, C. elegans; Dm, D. melanogaster; Hs, H. sapiens; Mm, M. musculus; Rn, R. norvegicus; Sc, S. cerevisiae; Sp, S. pombe. Accession numbers (p, protein; n, nucleotide): Ce-Fzo, p-AAC71095; Dm-FzoS, n-U95821; Dm-FzoU, p-AAF46162; Hs-Mfn1, n-AF329637; Hs-Mfn2, n-D86987; Mm-Mfn1, n-AK018181; Mm-Mfn2, n-AF384100; Rn-Mfn2, n-U41803; Sc-Fzo, n-Z36048; Sp-Fzo, p-CAA19004.

 


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  		Fig. 2. Mitofusin 2 is a ubiquitous mitochondrial protein. (A) Domain organization of mitofusin 2 (Mfn2) and of Mfn2-fragments (NG, CT) used for antibody generation. GB, GTP-binding; CC1, CC2, coiled-coil; TM, transmembrane domain; H, 6His-tag. (B) Western blots of subcellular fractions from mouse C2 and human Hela cells: TH, total homogenate; Mit, mitochondria; Cyt, cytosol; Mic, microsomes. The Mfn2-specific antiserum decorates a polypeptide (arrowhead) that is highly enriched in mitochondria, and whose apparent molecular mass is similar to that predicted for Hs-Mfn2 (86.4 kDa). The distribution of the adenine nucleotide translocator (ANT) confirms mitochondrial enrichment. The position and size (in kDa) of molecular mass markers is indicated. (C) Western blot analysis of mitochondrial fractions from human HeLa (He) and mouse C2 cells (C2) and from rat liver (L), kidney (K), brain (B) and muscle (M) with affinity purified antibodies specific for Mfn2-fragments NG or CT. (D) Double immunofluorescence on human skin fibroblasts with antibodies against the mitochondrial marker COX4 and against an N-terminal fragment (NG) of Mfn2. Mfn2 and COX4 colocalize in mitochondria. Bar, 15 µm.

 


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  		Fig. 3. Membrane topology of mitofusin 2. (A) Structure of myc-tagged (M) Mfn2 (MF2) and Mfn2-mutants (MF2-IYFFT, MF2-NT, MF2-TMCT). (B) Antibody specificity. Cells were transfected with myc-tagged mitofusins (MF1, MF2) and Mfn2-fragments (MF2-IYFFT, MF2-NT, MF2-TMCT) and analyzed by western-blot with the indicated antibodies (myc, NG, CT). The images visualize overexpressed proteins at exposures where endogenous Mfn2 is hardly visible (NG, CT). Myc-specific antibodies decorate both mitofusins and the Mfn2-constructs (arrows). NG and CT do not decorate Mfn1 and are specific for their respective Mfn2-domains (arrows). (C,D) Western blot analysis of isolated mitochondria treated with Proteinase K for the indicated times (minutes) in the absence (C) or presence (D) of Triton X-100. Proteins of the intermembrane space (Cyt.c) and of the mitochondrial matrix (Hsp60) are protected in intact mitochondria (C) and are degraded after membrane solubilization (D). In the absence of detergent (C), Mfn2 is gradually degraded into fragments (asterisks) that are differentially recognized by domain-specific antibodies (NG, CT). These fragments are smaller than MF2-NT/MF2IYFFT (upper arrow) and MF2-TMCT (lower arrow), respectively. This reveals the cleavage of N-terminal and C-terminal domains and their exposure towards the protease-containing solution. The position and size (in kDa) of molecular mass markers is indicated by arrowheads. (E) Deduced membrane topology of Mfn2. OM and IM, outer and inner mitochondrial membranes.

 


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  		Fig. 4. Involvement of the C-terminal and of the coiled-coil domains in mitochondrial targeting. (A) Structure and localization (Loc.) of myc-tagged mitofusins and Mfn2-constructs. mt, mitochondria; ct, cytoplasma; ER, endoplasmic reticulum. (B-F) Cells were transfected with mitochondrial GFP (mtGFP) and/or with the indicated Mfn2-constructs. Myc-tagged Mfn2-mutants (myc) and the ER-marker calnexin were visualized with specific antibodies. (B) MF2-TMCT colocalizes with mtGFP in mitochondria. (C) MF2-IYFFT colocalizes with calnexin in the ER. (D-F) MF2-RRD, MF2-{Delta}C1 and MF2-{Delta}C2 are targeted to mitochondria, albeit inefficiently. A fraction of these molecules is mistargeted, especially at high expression levels. Bars, 15 µm.

 


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  		Fig. 5. The coiled-coil domains of Mfn2 interact with each other and mediate mitochondrial localization of mistargeted Mfn2-fragments. (A-D) HeLa cells were co-transfected with plasmids encoding Myc-tagged Mfn2-fragments (MF2-NT, MF2-IYFFT) and with a plasmid encoding the transmembrane and C-terminal domain of Mfn2 (F2-TMCT). Molecules were visualized with antibodies against myc and CT, respectively. (A) Soluble Mfn2-fragments truncated before the transmembrane domain (MF2-NT) localize to mitochondria upon co-expression of the transmembrane and C-terminal domains of Mfn2 (F2-TMCT). (B) MF2-NT molecules devoid of their coiled-coil domain (MF2-NT{Delta}C1) remain cytosolic despite the co-expression of F2-TMCT. (C,D) Mfn2-mutants normally targeted to the endoplasmic reticulum (MF2-IYFFT) localize to mitochondria upon co-expression of F2-TMCT. (D) Co-expressed Mfn2-fragments can redistribute and cluster mitochondria in the perinuclear region. (E) Cells transfected with the indicated plasmids were processed for immunofluorescence and the localization of MF2-NT or MF2-IYFFT was determined. Where indicated ({Delta}C1, {Delta}C2), molecules were devoid of their coiled-coil domains. The results are expressed as percentage of transfected cells in which MF2-NT or MF2-IYFFT localize to mitochondria (n>=200 cells for each category). The mitochondrial relocalization of MF2-NT and MF2-IYFFT by F2-TMCT depends on the presence of coiled-coil domains on both Mfn2-fragments. Bars, 15 µm.

 


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  		Fig. 6. Excess Mfn2 redistributes and clusters mitochondria in a process independent of the cytoskeleton. Immunofluorescence analysis of untransfected HeLa cells (A,B) and of Hela cells expressing mtGFP and/or Mfn2 (C,D). (A) The distribution and morphology of elongated mitochondria (COX2) is mildly perturbed upon deplymerazation of microtubules (tub) with nocodazole. (B) Addition of cytochalasin B (cytochalasin) leads to the dissappearance of elongated stress fibers (act) and to the appearance of punctated mitochondria scattered throughout the cell (COX2). Both drug treatments lasted 2 hours. (C,D) Mfn2-overexpression leads to mitochondrial redistribution and clustering (mtGFP, COX2) in the presence (control) and absence (nocodazole, cytochalasin) of a tubulin or actin cytoskeleton. Drug treatments started 9 hours after transfection, before significant expression of exogenous Mfn2. Cells were processed for immunofluorescence after a further 15 hours. Bars, 15 µm.

 


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  		Fig. 7. Excess Mfn2 modifies mitochondrial morphology. HeLa cells were transfected with expression vectors encoding mtGFP (A, control), mtGFP and Mfn2 (A, +Mfn2) or MF2-IYFFT (B,C). Cells were fixed and decorated with specific antibodies directed against an inner membrane marker (COX2), calnexin or the myc-epitope (myc). Cells were visualized by confocal (A) or conventional microscopy (B,C). The images depict selected cell regions (A) or entire cells (B,C). The insets show enlargements of the areas marked with arrowheads. (A) In control cells, mtGFP and COX2 colocalize in mitochondrial tubules of several µm length and 400-450 nm diameter. In Mfn2 overexpressing cells, mitochondria are spherical (mean diameter, 860±160 nm, n=34) and clustered. In enlarged spherical mitochondria, markers of the inner membrane (COX2) appear to surround the matrix space (mtGFP). (B) At high expression levels, MF2-IYFFT (myc) segregates within the ER (calnexin) and forms membrane networks with modified morphology. (C) MF2-IYFFT expression (myc) does not affect mitochondrial morphology and distribution (mtGFP). Bars, 5 µm (main images) and 1 µm (insets).

 


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  		Fig. 8. The ultrastructure of clustered mitochondria is modified and their enveloping double membranes are closely apposed. Hela cells were transfected with an empty vector (A) or with a vector encoding Mfn2 (B,C) and processed for electron microscopy. (A) In control cells, sections depict tubular mitochondria of 250-350 nm diameter.

(B) Mitochondrial distribution and morphology is modified in Mfn2- transfected cells. Mitochondria are clustered, and their diameters can surpass 1 µm (upper left region). In enlarged mitochondria, cristae membranes are swollen and/or difficult to identify. (C) The double membranes of clustered mitochondria are in direct contact (arrowheads), but do not merge. Arrows in B and C point to electron dense material that is absent from control cells, and accumulates between mitochondria. Bars, 500 nm (A,B); 200 nm (C).

 




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