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First published online 28 June 2005
doi: 10.1242/jcs.02449

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Mutational analysis of action of mitochondrial fusion factor mitofusin-2

Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

View larger version (69K): [in a new window]   Fig. 1. Comparison of the sequences of Fzo/Mfn and structure of mammalian Mfn2. (A) The sequences were aligned by the ClustalW program. A black or gray background indicates that amino acid residues of all or four of the five highly homologous proteins are identical, respectively. m, Mus musculus; d, Drosophila melanogaster; c, Caenorhabditis elegans; TM, transmembrane. (B) Domain structure of mouse Mfn2. C, predicted coiled-coil domain; gray, GTPase domain; black, predicted transmembrane domains. Numbers indicate amino acid residue positions at the beginning and end of domains. Vertical lines in the lower scheme represent conserved amino acid residues. The GTPase domain is highly conserved among species. The GenBank accession numbers of the proteins are: mMfn1 (NP_077162), mMfn2 (AAH46503, dMfn (AAF46161, cFzo (T34496) and dFzo (AAC24457.

View larger version (49K): [in a new window]   Fig. 2. Segments R1, R3, R6 and R7, and GTPase activity of Mfn2TM are necessary for mitochondrial fragmentation in COS7 cells. (A) Schematic diagram of the Mfn2 deletion and point mutants used to analyze mitochondrial fragmentation, and percentage of transfected cells with fragmented mitochondria. All constructs are epitope-tagged with the 3 xFLAG sequence present at the N terminus. Boxed C, predicted coiled-coil domain; gray, GTPase domain; black, predicted transmembrane domains. The G2 Mfn2-T130A mutation is equivalent to a mutation in the Ras GTPase that abolishes effector interactions. (B) COS7 cells were transiently transfected with expression vector for FLAG-Mfn2TM (a,b) and FLAG-Mfn2R4/R5/TM (c,d). After 16 hours, the cells were labeled with MitoTracker to visualize mitochondria (b,d), fixed, and stained with anti-FLAG antibody followed by Alexa Fluor 488-conjugated secondary antibody (a,c). The star indicates untransfected cell with normal reticular mitochondria. Bar, 20 µm.

View larger version (44K): [in a new window]   Fig. 3. GTPase-dependent interaction of the Mfn2 amino and carboxyl termini exposed to cytoplasm is necessary for mitochondrial fragmentation in COS7 cells. (A) Schematic diagram of the Mfn2 deletion and point mutants used to analyze mitochondrial fragmentation, and percentage of transfected cells with fragmented mitochondria. All constructs of Mfn2 with the N terminus exposed to the cytoplasm were epitope-tagged with the 3 xFLAG sequence present at the C terminus. Mfn2C construct is epitope-tagged with the Myc sequence present at the N terminus. Boxed C, predicted coiled-coil domain; gray, GTPase domain; black, predicted transmembrane domains. The G2 Mfn2-T130A mutation is equivalent to a mutation in the Ras GTPase that abolishes effector interactions. (B) COS7 cells were transiently cotransfected with expression vectors for Myc-Mfn2C and Mfn2N-FLAG (a-f) or Mfn2NT130A-FLAG (g-i). After 16 hours, the cells were labeled with MitoTracker to visualize mitochondria (c,f,i), fixed, and stained with anti-FLAG antibody and anti-Mfn2C antibody followed by Alexa Fluor 488-conjugated secondary antibody (a,d,g) and Alexa Fluor 350-conjugated secondary antibody (b,e,h), respectively. Mitochondrial fragmentation was induced in COS7 cells expressing both Mfn2N and Mfn2C (arrow) but not Mfn2N alone (star; reticular structure is normal) or Mfn2C alone (arrowhead). Faint mitochondrial staining, seen in b, e and h, is not due to staining of endogenous Mfn2 on mitochondria by anti-Mfn2C since it disappeared when Alexa Fluor 350-labeled secondary antibody (blue) was replaced with Alexa Fluor 488-labeled one (green). The staining is probably an artifact caused by autofluorescence: on blue laser irradiation, mitochondria become weakly autofluorescent (blue). Fluorescence of Alexa Fluor 350 is relatively weak compared to that of Alexa Fluor 488 and requires longer exposure for imaging. Under such conditions, mitochondrial autofluorescence becomes inevitable. (C) HEK293T cells were transiently cotransfected with expression vectors for Myc-Mfn2C and Mfn2N-FLAG, Mfn2NT130A-FLAG, Mfn2NR2-R4-FLAG, Mfn2NR1-FLAG, or Mfn2NR1/GTPase-FLAG. After 16 hours, cell lysates were immunoprecipitated with anti-FLAG affinity agarose, separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with rabbit anti-Mfn2C antiserum or anti-FLAG antibody. Bar, 20 µm.

View larger version (43K): [in a new window]   Fig. 4. Loss of mitochondrial membrane potential induced by overexpression of the fusion protein lacking the GTPase domain and transmembrane domain of Mfn2, in COS7 cells. (A) Schematic diagram of the Mfn2 deletion mutants used and percentage of transfected cells with loss of mitochondrial membrane potential. All constructs are epitope-tagged with the 3 xFLAG sequence. Yellow, predicted coiled-coil domains; gray, GTPase domain; black, predicted transmembrane domains. (B) COS7 cells transiently transfected with expression vector for FLAG-Mfn2GTPase/TM (a,b) and FLAG-Mfn2R2/R6 (c,d). After 16 hours of transfection, the cells were stained by the membrane potential-sensitive dye MitoTracker Red (b,d), fixed, and then immunostained with anti-FLAG antibody followed by Alexa Fluor 488-conjugated secondary antibody (a,c). An arrow and an arrowhead indicate transfected cells expressing FLAG-Mfn2GTPase/TM and FLAG-Mfn2R2/R6, respectively; stars indicates normal mitochondria with a reticular network. (C) COS7 cells transiently transfected with an expression vector for FLAG-Mfn2GTPase/TM (a-d). After 16 hours of transfection, the cells were stained with MitoTracker as the membrane potential-sensitive dye (b), fixed, and stained with anti-Mfn2-C antibody (a) and anti-Hsp60 antibody as mitochondrial marker (c) followed by Alexa Fluor 350-conjugated secondary antibody and Alexa Fluor 488-conjugated secondary antibody, respectively. A merged image is shown in panel d. (D) Mfn2GTPase/TM-induced loss of mitochondrial membrane potential without loss of mitochondrial DNA. COS7 cells expressing Mfn2GTPase/TM were stained with MitoTracker, with Hoechst 33342 as DNA dye (b), and anti-Hsp60 antibody as mitochondrial marker (k) followed by Alexa Fluor 488-conjugated secondary antibody. Merged images are shown in a and d; (b-d) higher magnifications of the boxed area in a. Bar, 20 µm.

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