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Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin

Abstract

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology1,2. Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy2,3,4. However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.

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Figure 1: Preparation of recombinant human OPA1 proteins using silkworm larvae.
Figure 2: L-OPA1-dependent membrane fusion.
Figure 3: Heterotypic membrane fusion via opposed L-OPA1 and CL.
Figure 4: Specific L-OPA1 to CL interaction is required for membrane fusion.
Figure 5: Role of S-OPA1 in L-OPA1-mediated membrane fusion.

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Acknowledgements

We thank J. Mima (Osaka University) for advice on the preparation of assays with proteoliposomes, K. Tabata (Kyushu University) for advice on the silkworm expression system, Kurume University EM laboratory, and H. Yagi (Tottori University) and D. Ozawa (Osaka University) for helping with the EM observations, and W. Nishihira for technical support. This work was supported by JSPS KAKENHI grant numbers 26291044 and 16H01209 (N.I.), and 26840026 (T.B.), MEXT-Supported Program for the Strategic Research Foundation at Private Universities, the Takeda Science Foundation (N.I.), the Ono Medical Research Foundation (N.I.) and the Ishibashi Foundation (T.B.).

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Contributions

N.I. and T.B. designed the research. T.B., T.I., H.K., S.S. and A.I. performed the experiments. K.Maenaka established and provided the bacmid expression vector system, and T.O. prepared basic procedures for expression and reconstitution of membrane proteins. K.Mihara supervised the study. N.I. and T.B. wrote the manuscript with the comments of the other authors.

Corresponding author

Correspondence to Naotada Ishihara.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Preparation of recombinant OPA1 and OPA1-dependent fusion in vitro or in vivo.

(a) Procedures for the fractionation and purification of recombinant OPA1. (b,c) SDS-PAGE analysis and Coomassie blue staining of L-OPA1 (b) and S-OPA1 (c). Asterisk: OPA1 protein. Fractionation experiments were carried out more than 10 times for L-OPA1 and more than 3 times for S-OPA1 with similar results. (d) Calculation of fusion activity by fitting to the exponential curve with the trace from the experiments shown in Fig. 2a. In the 1:4,000 condition, 25 L-OPA1 molecules, which might form 2.5 complexes (Fig. 1h) in each liposome (the number of lipid molecules 100,000)35, could mediate membrane fusion. The goodness of fit was evaluated by the R2 value. (e) Preparation of the OPA1 KO cell line using the CRISPR/Cas9 system. Whole cell homogenates from OPA1 WT or KO HeLa cells were prepared and subjected to immunoblot analysis with the indicated antibodies (the experiment was performed one time). (f) OPA1 WT or KO HeLa cells were fixed, permeabilized, subjected to immunofluorescence analysis with antibodies to TOM20 (red), and examined by confocal microscopy (representative images of more than 3 independent experiments with similar results). Magnified images are shown in the boxed regions. Scale bars, 10 μm. (g,h) Heterotypic mitochondrial fusion in HeLa cells. Mitochondrial fusion was analyzed in cell hybrids from cells expressing mitGFP and mitRFP using OPA1-deficient or control HeLa cells. After 6 h culture, fusion efficiency was analyzed by fluorescence microscopy (g) and cell counting (h). n, number of cell hybrids analyzed from 3 independent experiments: WT × WT (n = 30), WT × CRISPR-OPA1 (n = 30), and CRISPR-OPA1 × CRISPR-OPA1 (n = 30). Scale bar: 10 μm. Statistics source data are available in Supplementary Table 1. Unprocessed original scans of blot is shown in Supplementary Fig. 3.

Supplementary Figure 2 CL conditions for regulation of mitochondrial morphology and quality control.

(a) Comparison of the number, length, and unsaturation of acyl chains of CL used in this study. (b) GTP hydrolysis activity of the deletion mutant (Δ581–941) of L-OPA1 (n = 3 independent measurements, data are mean ± s.d.). (cg) KD of CLS1 using siRNA. (c) RNAs were analyzed by RT-PCR (the experiment was performed one time). Nuclear genome-encoded transcripts were analyzed. (d) Fluorescence images of the control and CLS1 KD cells (representative images of more than 3 independent experiments with similar results). Scale bar: 10 μm. (e) Immunoblot analysis of mitochondrial protein levels using the indicated antibodies (the experiment was performed one time). (f,g) Mitochondrial fusion in CLS1 KD cells and OPA1 KD cells. Mitochondrial fusion was analyzed using HeLa cells expressing mitGFP and mitRFP that were co-cultured and fused with the HVJ envelope. After 6 h culture, fusion efficiency was analyzed by fluorescence microscopy. n, number of cell hybrids analyzed from 3 independent experiments: cont × cont (n = 38), CLS1KD × CLS1KD (n = 41), and OPA1KD × OPA1KD (n = 32). Scale bar: 10 μm. Statistics source data are available in Supplementary Table 1. Unprocessed original scans of agarose gel and blots are shown in Supplementary Fig. 3. (h) Model of the isolation of damaged mitochondria from the active mitochondrial network. OPA1 inactivation and CL remodeling are used as markers for the specific selection of damaged mitochondria.

Supplementary Figure 3 Unprocessed original scans of Coomassie-stained gels, agarose gels, and immunoblots shown in the Figures and Supplementary Figures.

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Ban, T., Ishihara, T., Kohno, H. et al. Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat Cell Biol 19, 856–863 (2017). https://doi.org/10.1038/ncb3560

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