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. 2017 Nov 14;114(46):E9863-E9872.
doi: 10.1073/pnas.1708782114. Epub 2017 Nov 1.

Sequences Flanking the Transmembrane Segments Facilitate Mitochondrial Localization and Membrane Fusion by Mitofusin

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Free PMC article

Sequences Flanking the Transmembrane Segments Facilitate Mitochondrial Localization and Membrane Fusion by Mitofusin

Xiaofang Huang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion.

Keywords: endoplasmic reticulum; membrane fusion; membrane targeting; mitochondria; mitofusin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Localization of MFN-ATL chimeras. (A) The domain structures of human ATL1 and MFN1 are shown in yellow and cyan, respectively. The first letter of each domain label represents the molecule (A for ATL1 and M or MFN1), and the following letters represent the domain type (CT, C-terminal tail; G, GTPase; HB, helix bundle; TM, transmembrane segments). The residue numbers used in constructing the chimeras are listed, and the predicted HR1 and HR2 of MFN1 are highlighted. hs, Homo sapiens. (B) HA-tagged chimeras that contain the ATL-TM domain were expressed in COS-7 cells. Localization was investigated using anti-HA antibodies (green) and compared with endogenous luminal ER protein, calreticulin (blue), or MitoTracker (red) by indirect immunofluorescence and confocal microscopy. The domain structures of the indicated chimeras are shown above the images. (C) As in B, but with cells expressing HA-tagged chimeras containing the MFN-TM domain. The Insets in B and C show the 2× enlargement of the indicated areas. (Scale bar for B and C: 10 μm.)
Fig. 2.
Fig. 2.
Functional analysis of MFN-ATL chimeras in yeast cells. (A) A GFP fusion protein containing the ER protein Sec63p was expressed in yeast cells lacking Sey1p and Yop1p (sey1Δyop1Δ cells). The ER morphology represented by the Sec63p-GFP signal was visualized by confocal microscopy, with the microscope focused on either the center or the periphery of the cell. ER morphology was categorized into three classes. Note that the partially abnormal class exhibits some fenestrated ER but is void of ER in some areas at the periphery of the cell. (Scale bar: 1 μm.) (B) Empty vector or indicated chimeras were expressed with a CEN plasmid under the control of the endogenous SEY1 promoter in sey1Δyop1Δ cells. See Fig. S5 for expression levels and localization of the chimeras. The ER morphology was determined as in A, and each category was colored as in A. A total of 80 to 150 cells were categorized for each sample. The experiments, performed in a blinded manner, are averages of three repetitions. (C) The same chimeras as in B were transformed into ufe1-1sey1Δ, and empty vector was transformed into ufe1-1 or ufe1-1sey1Δ. Serial 10-fold dilutions of cells were spotted onto an SC medium and incubated at 30 °C for 3 to 4 d. (D) Growth rates of the same cells as in C were determined by measuring OD600.
Fig. 3.
Fig. 3.
An amphipathic helix in the MFN1-CT. (A) Sequence alignment of the CTs from various MFNs. The predicted and observed α-helices of MFN1 are labeled with cylinders. The sequences of three synthetic peptides used in this figure are underlined in red. Mutated residues are numbered, with critical ones in cyan, moderately critical ones in green, and noncritical ones in yellow. The conservation of the important residues is highlighted in black boxes. (B) Helical wheel representation of α10 was generated using program HeliQuest (heliquest.ipmc.cnrs.fr/). Hydrophobic, negatively charged, and positively charged residues are shown in yellow, red, and blue, respectively. Mutated residues are numbered. (C) Indicated peptides were added to liposomes with or without PC containing doxyl groups at position 5 or 12 of the hydrocarbon. The quenching of the fluorescence of Trp in the peptide was measured and expressed as F0/F (maximal fluorescence with doxyl-free liposomes divided by maximal fluorescence with doxyl-containing ones). DmATL CTH is a positive control, and amino acid Trp is a negative control. Data shown are the mean and SE of three experiments. (D) Circular dichroism spectra of indicated peptides were recorded in the absence (black lines) or presence (red lines) of liposomes. EPL, liposomes made of E. coli polar lipids; M.R.E., mean residue ellipticity; PCPS, liposomes made of PC:PS (mole percent 85:15). (E) HA-tagged WT MFN1 or indicated mutants were transfected into MFN1-deleted MEF cells. Their localization was determined by anti-HA antibodies (green) and compared with that of MitoDsRed, a mitochondrial targeted marker protein, using indirect immunofluorescence and confocal microscopy. The right panels show the 4.7× enlargement of the indicated areas. (Scale bars: 10 μm.) (F) The mitochondrial morphology of indicated samples was categorized as “normal” or “abnormal”. A total of 100 to 120 cells were counted for each sample. All graphs are representative of at least three repetitions. (G) Full-length dmATL, ATL tailless, tailless-CTMα10, and tailless-CTMα11 were purified and reconstituted into donor and acceptor vesicles at a 1:2,000 protein-to-lipid ratio. GTP-dependent fusion of donor and acceptor vesicles was monitored by the dequenching of an 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled lipid present in the donor vesicles. All reactions were initiated by addition of GTP. (H) As in G, with indicated constructs tested at a 1:1,000 protein-to-lipid ratio.
Fig. 4.
Fig. 4.
TM-flanking sequences that support MFN1 targeting. (A) COS-7 cells were transfected with HA tagged WT MFN1 or indicated mutants. Their localization was determined by anti-HA antibodies (green) and compared with that of MitoDsRed using indirect immunofluorescence and confocal microscopy. (Scale bar: 10 μm.) (B) The membrane interacting regions of BDLP (PDB ID code 2W6D) are shown in schematic representation. Lipids are shown in yellow sticks. (C) Sequence alignment of the loop between α7 and α8. The conserved residues are highlighted with black boxes. (Right) The helical wheel representation of the loop generated using HeliQuest. (D) As in A, but with cells expressing Myc-tagged mutants in the α7 and α8 loop. Their localization was determined by anti-Myc antibodies (green) and compared with that of Tom20 (red), a mitochondrial membrane protein, using indirect immunofluorescence and confocal microscopy. (Scale bar: 10 μm.) (E) Domain functions of MFN. MFN is shown in cyan. The proposed function of each domain is indicated. HB, helix bundle; HR2, heptad repeat 2; TM, transmembrane domain; α7α8, loop connecting α7 and α8.

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