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First published online 28 June 2005
doi: 10.1242/jcs.02415

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The mitochondrial protein MTP18 contributes to mitochondrial fission in mammalian cells

Daniel Tondera^*, Frank Czauderna, Katharina Paulick, Rolf Schwarzer, Jörg Kaufmann and Ansgar Santel

Atugen AG, Robert-Rössle-Str.10, Otto-Warburg-Haus (80), 13125 Berlin, Germany

View larger version (81K): [in a new window]   Fig. 1. Drp1-mediated fission in MTP18-myc transfected cells. High magnification confocal microscopy images of mitochondria from MTP18-myc transfected COS-7 cells. Images show overview and close-up view of mitochondrial morphology (anti-MTP18, red) and Drp1 localisation (arrows, anti-Drp1, green) before (A) and after fragmentation (B). Double-arrows indicate mitochondrial constrictions at sites were Drp1 localises.

View larger version (57K): [in a new window]   Fig. 2. MTP18, a putative internal mitochondrial TM protein. (A) Hydrophobicity plot for MTP18 as measured by the TMpred algorithm showing probability for TM domains. (B) Western blot of mitochondrial extracts treated with Na2CO3 and NaCl after separation by ultracentrifugation in soluble (S, peripheral membrane proteins) and pelleted proteins (P, integral membrane proteins). Blots were probed with anti-Hsp60, anti-hFis1 and anti-MTP18. (C) Protease digestion of mitochondria with proteinase K (PK) or trypsin (lanes 2 and 4, or 3, respectively). Extracts were subjected to SDS-PAGE and western blots were carried out with anti-MTP18, anti-Mfn1, anti-cytochrome c and anti-Hsp60. Mitochondria were also osmotically shocked to disrupt their outer membrane and treated with PK (lane 4). MTP18 becomes degraded like cytochrome c upon osmotic shock. (D) Confocal microscopy of mitochondria from MTP18-myc transfected COS-7 cells stained with anti-MTP18 (green) and anti-Hsp60 (red). MTP18-myc did not completely colocalise with the mitochondrial matrix protein Hsp60. Arrows indicate arcas of mitochondria where MTP18 does not colocalise with Hsp60; double arrow indicates mitochondria from an untransfected cell.

View larger version (52K): [in a new window]   Fig. 3. Effect of the MTP18 C-terminus on protein localisation and mitochondrial morphology. (A) (left) diagram of myc-tagged (C-terminal red boxes) MTP18 wild-type and mutants that lack the N-terminus (including the predicted TM1) or the C-terminus (including the predicted TM2 and TM3). Transient protein expression was confirmed by western blot (right panel) probed with anti-MTP18 (*, endogenous MTP18; arrow, full-length MTP18–myc; arrowhead, truncated protein variants. (B) Representative confocal immunofluorescence images of transiently transfected COS-7 cells expressing wild-type (upper row), N-terminally truncated (middle row) and C-terminally truncated MTP18 (lower row). Shown is distribution of MTP18 (green, anti-MTP18) and mitochondrial morphology (red, anti-Hsp60). Bars, 20 µm. (C) Immunofluorescence staining of 91-166-MTP18-myc in transfected COS-7 cells with anti-MTP18 (green) and anti-Hsp60 (red) to investigate its subcellular localisation. Arrowheads, untransfected cells; arrow, cell with low expression levels of 91-166-MTP18-myc; double-arrow, cell with high expression levels of 91-166-MTP18-myc.

View larger version (52K): [in a new window]   Fig. 4. MTP18-induced mitochondrial fission is inhibited by counteracting fusion activity. (A) Representative picture of a COS-7 cell transiently expressing MTP18-myc (red) and the mitochondrial adenosine nucleotide transporter as a GFP-fusion protein (ANT-GFP, green). The merged picture shows fragmented mitochondria (yellow). (B) Western blot with extracts from COS-7 cells that had been transiently transfected with MTP18, Drp1 or Mfn1 expression constructs. Endogenous and recombinant proteins were detected with appropriate antibodies. Arrow, MTP18-myc; double arrow, GFP-Mfn fusion protein; arrowheads as labelled. (C) COS-7 cell transfected with dominant negative HA-Drp1K38A alone (green, anti-Drp1; red, Mitotracker) or together with MTP18-myc (MTP18-myc, red). (D) Representative confocal microscopy images of COS-7 cells transiently transfected with GFP-Mfn1 alone (mitochondrial morphology shown with Mitotracker dye) or together with MTP18-myc, or with GFP-Mfn1K88T and MTP18-myc. MTP18 was detected with anti-MTP18 (red); GFP-Mfn1 fusion protein is shown in green. (E) Perecentage of transfected cells that exhibit indicated mitochondrial morphologies.

View larger version (55K): [in a new window]   Fig. 5. RNAi-mediated reduction of MTP18 results in mitochondrial fusion. Two examples of specific RNAi-mediated protein reduction is shown on western blots with extracts from HeLa cells transiently transfected with shRNA-expressing plasmids (as indicated) to target MTP18, Mfn1, Drp1 and p110; hFis1 served as loading control. (B) Representative confocal images of HeLa cells (right panels, higher magnification) transiently transfected with indicated shRNA-expressing vectors labelled with anti-Hsp60 and treatment with nocodazole before staining to reveal mitochondrial morphology. Bars, 20 µm. Diagrams show the percentage of transiently transfected HeLa cells (corresponding to the cell shown on the left) with indicated mitochondrial morphology (Fu, highly fused mitochondria; Nw, filamentous mitochondrial network; Fi, highly fragmented mitochondria). The majority of cells that show highly fused mitochondria were found in cells expressing shRNA against MTP18 and Drp1. Fragmented mitochondria were found in cells expressing shRNA against Mfn1. No obvious mitochondrial phenotype was found in cells expressing p110-shRNA.

View larger version (54K): [in a new window]   Fig. 6. hFis1 mitochondrial fission activity depends on MTP18. (A) HeLa cells were transiently transfected with expression constructs for shRNA-mediated RNAi (shRNA-hFis1, lane 2) or combined with protein overexpression (shRNA-hFis1/MTP18-myc or shRNA-MTP18-myc/hFis1, lane 3 or 4 respectively). Corresponding protein extracts were subjected to SDS-PAGE and the amount for endogenous and recombinant proteins was assessed by western blot. Mfn1 protein levels served as loading control. (B) RNAi for hFis1 gave rise to highly fused mitochondria in shRNA-expressing, transiently transfected HeLa cells as revealed by anti-cytochrome c staining (representative picture, upper left panel; higher magnification, right panel). Cells were treated with nocodazole before fixation and immunofluorescence staining. Overexpression of hFis1 resulted in fragmentation of mitochondria (second row of images) as shown by immunofluorescence staining with anti-hFis1 (green) – to identify HeLa cells expressing high levels of hFis1 – and anti-cytochrome c (red) to assess mitochondrial morphology (arrow indicates an untransfected cell). The third and fourth panel show merged images with hFis1 and cytochrome c colocalisation and a close-up view of fragmented mitochondria. (C) HeLa cells were doubly transfected with MTP18-myc expression construct and shRNA-Fis1 RNAi vector in order to target hFis1. Cells with high levels of MTP18 were identified by immunofluorescence using anti-MTP18 (green). Mitochondria were visualised by labelling with anti-cytochrome c (red). High levels of MTP18 resulted in fragmented mitochondria (double arrow). By contrast, cells that do not transiently express of MTP18-myc show fused mitochondria due to RNAi of hFis1 RNA (arrow). Overexpression of MTP18 led to fragmented mitochondria in cells with reduced hFis1 levels (merged higher magnification image on right). (D) Reduced levels of MTP18 abolished hFis1-mediated mitochondrial fragmentation after transient overexpression. HeLa cells doubly transfected with shRNA-MTP18 and hFis1 expression constructs are shown. hFis1 was detected by immunofluorescence with anti-hFis1 (green). Arrows indicates cells with endogenous levels of hFis1; double-arrows points to cells transiently expressing high levels of hFis1. Mitochondrial morphology was revealed by immunostaining with anti-cytochrome c. High levels of hFis1 had no effect on the fused mitochondrial morphology due to loss of MTP18 by RNAi (merged image and higher magnification, last two panels on right). Bars in the first three panels of the second row in B, and in C and D, 20 µm. (E) Percentage of doubly transfected HeLa cells (RNAi in combination with overexpression) that show the indicated mitochondrial morphology. Fu, mainly fused mitochondria; Nw, network of filamentous mitochondria; Fi, mitochondria with highly fragmented appearance. RNAi against hFis1 (green bars) together with overexpressed MTP18; RNAi for MTP18 together with overexpressed hFis1 (red bars).

View larger version (36K): [in a new window]   Fig. 7. Knockdown of Drp1 affects MTP18-induced mitochondrial fission. (A) HeLa cells were co-transfected with an shRNA-Drp1 expression plasmid for RNAi and with an MTP18-myc expression vector. The resulting knockdown of Drp1 together with overexpression of MTP18-mycis was shown by western blotting. No changes in protein levels were detected for hFis1 and Mfn1. Cells were treated with nocodazole and stained with anti-MTP18 (green) to show recombinant MTP18-myc overexpressing cells (arrows indicate representative examples) and anti-cytochrome c (red) to show mitochondrial morphology. Mitochondria show highly fused morphology (merged picture and high magnification, two panels on right) due to RNAi-induced reduction of Drp1 in cells with endogenous levels of MTP18 (double-arrow; not detectable with anti-MTP18) as well as in cells with high levels of MTP18-myc (arrow).

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