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. 2014 Mar 17;204(6):919-29.
doi: 10.1083/jcb.201308006. Epub 2014 Mar 10.

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Ruchika Anand  1 , Timothy Wai, Michael J Baker, Nikolay Kladt, Astrid C Schauss, Elena Rugarli, Thomas Langer
Affiliations

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Ruchika Anand et al. J Cell Biol. .

Abstract

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER-mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.

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Figures

Figure 1.
Figure 1.
Loss of OMA1 restores tubular mitochondria in Yme1l−/− cells. (A) Representative images of mitochondrial morphology in MEFs. The asterisks denote the area of magnification enlarged on the right. Bars: (left) 15 µm; (right) 5 µm. (B) Quantification of three independent experiments (error bars indicate mean ± SD), n ≥ 100. (C) Accumulation of OPA1 forms in MEFs lacking YME1L, OMA1, or both. Cells lysates were analyzed by SDS-PAGE analysis and immunoblotting using the indicated antibodies. a–e, OPA1 forms. (D) Schematic representation of mature L-OPA1 forms derived from splice variants 1 and 7 and S-OPA1 forms produced by cleavage at proteolytic sites S1 or S2 by OMA1 or YME1L, respectively.
Figure 2.
Figure 2.
OPA1 processing is dispensable for mitochondrial fusion. (A and B) Photoactivatable GFP (matrix-PA-GFP) and mito-mCherry both targeted to the mitochondrial matrix were expressed in WT, Yme1l−/−Oma1−/−, and Opa1−/− MEFs (A). Fusion was monitored by the time-dependent dilution and redistribution of PA-GFP fluorescence (top, 1 min; bottom, 60 min). Bars, 15 µm. (B) Quantification of mitochondrial fusion. Results are represented in the form of a box plot: boxes represent data between the 25th and 75th percentiles, lines extend between the 10th and 90th percentiles, the horizontal line indicates the median, and the circle indicates the mean value (n ≥ 14; Yme1l−/−Oma1−/− vs. WT, P = 0.8; Yme1l−/−Oma1−/− vs. Opa1−/−, P = 1.6 × 10−12; WT vs. Opa1−/−, P = 4.6 × 10−8; ***, P ≤ 0.001). (C) Visualization of individual fusion events in Yme1l−/−Oma1−/− MEFs using matrix-PA-GFP and mito-mCherry. The arrowheads indicate mitochondria that have acquired photoactivated GFP due to fusion with photoactivated mitochondria. Bar, 5 µm. (D) Mitochondrial morphology in Opa1−/− MEFs expressing cleavable or noncleavable OPA1 forms. Flag-tagged variants of human OPA1 isoform 1 (S1-Flag), isoform 1 lacking the S1 site (ΔS1-Flag), or ΔS1-FlagK301A were transiently expressed in Opa1−/− cells. The mitochondrial network was visualized using a TOM20-specific antibody. Bar, 15 µm. (E) Quantification of three independent experiments (error bars indicate mean ± SD), n ≥ 100.
Figure 3.
Figure 3.
Loss of OMA1 restores cristae morphology and apoptotic resistance of Yme1l−/− cells. (A) WT, Oma1−/−, Yme1l−/−, and Yme1l−/−Oma1−/− MEFs were analyzed by transmission electron microscopy. Bar, 1 µm. (B–D) WT and protease-deficient MEFs were exposed to H2O2 to induce apoptosis. (B) Immunoblot analysis of apoptotic marker proteins (cleaved PARP, cPARP; caspase 3, CASP). (C and D) Flow cytometry analysis of viable cells (Q3: 7-AAD−;APC−) and late apoptotic cells (Q2: 7-AAD+; APC+). *, P ≤ 0.05; ***, P ≤ 0.001. Error bars indicate mean ± SD.
Figure 4.
Figure 4.
The function of short OPA1 forms is linked to mitochondrial fission. (A) Mitochondria fuse in Yme1−/− cells. Mitochondrial fusion was analyzed as in Fig. 2 using matrix-PA-GFP. (B) Quantification of mitochondrial fusion in Yme1l−/− cells after 60 min. Results are represented in the form of a box plot (see Fig. 2). WT versus Yme1l−/− cells, P = 0.14. (C) Expression of S-OPA1 forms in Yme1l−/−Oma1−/− cells induces mitochondrial fragmentation. Mitochondrial morphology was assessed in Yme1l−/−Oma1−/− MEFs expressing Flag-tagged variants of rat S-OPA1-Flag or S-OPA1K301A-Flag (bottom) harboring a mutation in the GTPase domain of OPA1. (D) Quantification of three independent experiments (error bars indicate mean ± SD). n ≥ 100; **, P ≥ 0.01. (E) Mitochondrial fusion occurs in fragmented mitochondria of WT and Yme1l−/−Oma1−/− cells expressing S-OPA1-Flag. (F) Quantification of mitochondrial fusion after 60 min. Results are shown in the form of a box plot (see Fig. 2). Shown are WT/Yme1l−/−Oma1−/− cells versus WT/Yme1l−/−Oma1−/− cells expressing rat S-OPA1. Bars, 15 µm.
Figure 5.
Figure 5.
GTPase-inactive short OPA1 forms colocalize with sites of mitochondrial division. (A) S-OPA1K301A-Flag expressed in Yme1l−/−Oma1−/− cells assembles into punctae partially colocalizing with ER–mitochondria contact sites (i; a total of 90 spots in five different cells), endogenous DRP1 (ii; a total of 230 spots in five different cells), and endogenous MID49 (iii; a total of 186 spots in six different cells). Bars, 2 µm. (B) Line scan of ii and iii along the lines indicated in A. (C) S-OPA1K301A-Flag punctae coalesce at sites of mitochondria constriction. Mitochondria are visualized using mito-mCherry, the ER using GRP78-specific antibodies. Bar, 2 µm.
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