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. 2007 Aug 27;178(5):757-64.
doi: 10.1083/jcb.200704112. Epub 2007 Aug 20.

Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage

Lorena Griparic  1 , Takayuki Kanazawa, Alexander M van der Bliek
Affiliations

Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage

Lorena Griparic et al. J Cell Biol. .

Abstract

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.

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Figures

Figure 1.
Figure 1.
The loss of mitochondrial membrane potential is enough to induce Opa1 proteolysis. (A) Western blots of cells treated for increasing lengths of time with CCCP show a shift in Opa1 isoforms. Tim23 serves as a loading control. Lack of cleavage of caspase 9 shows that CCCP did not induce apoptosis. Staurosporine (STS)-induced cleavage serves as a positive control. Opa1 bands were labeled a, b, c, d, and e as previously described (Duvezin-Caubet et al., 2006; Ishihara et al., 2006; Olichon et al., 2007). (B) A 5.5-h incubation with CCCP causes mitochondria to fragment but does not cause the release of cytochrome c or Opa1 into the cytosol. The merged images show cytochrome c in red and Opa1 in green. Bar, 10 μm.
Figure 2.
Figure 2.
Effects of known mitochondrial proteases on Opa1 proteolysis. (A) Cells were transfected with siRNA for the different proteases. Scrambled oligonucleotides served as controls. PARL was reduced by 92%, Yme1 was reduced by 72%, and HtrA2/Omi was reduced by 99.4% on Western blots. Proteolysis was induced with CCCP (60 min). DMSO (solvent) served as a control. For Yme1 and PARL siRNA, proteolysis was also induced with staurosporine (3 h). Data are representative of three independent experiments. (B) Densitometry of Opa1 bands in Yme1 siRNA–transfected cells with or without CCCP. The histogram shows the relative intensity of bands. (C) PARL−/− cells show enhanced degradation but still possess the inducible cleavage of Opa1. Endogenous protein was detected with Opa1 antibody. Transfected isoforms were detected with a C-terminal myc tag. The two cleavage products of isoform 7 correspond to cleavage in exon 5 (S1) and exon 5b (S2). (D) Effects of Yme1 siRNA on isoform 1, 5, and 7 cleavage with or without CCCP induction and deletion of the S1 cleavage site. (E) Densitometry of the uninduced lanes (DMSO) in D. A plus symbol after Delta S1 indicates an S1 cleavage site deletion (Ishihara et al., 2006). A plus symbol after Yme1 indicates transfection with Yme1 siRNA.
Figure 3.
Figure 3.
Disruption of mitochondrial morphology by Yme1 siRNA. Mitochondria were detected with Mitotracker. (A) Cells transfected with scrambled oligonucleotides for Yme1. (B) A cell 2 d after transfection with Drp1 siRNA. (C and D) Cells 2 d after transfection with Yme1 siRNA. (E and F) Cells 3 d after transfection with Yme1 siRNA. (G) Cells 2 d after transfection with Opa1 siRNA. (H) Cells 2 d after cotransfection with Opa1 and Yme1 siRNA (see Fig. S3 A for expression levels). (I and J) Cells transfected with Opa1 exon 4b– and 5b-specific siRNA (see Fig. S2 for quantification of the morphological defects; available at http://www.jcb.org/cgi/content/full/jcb.200704112/DC1). Bar, 10 μm.
Figure 4.
Figure 4.
Recovery of Yme1 siRNA–transfected cells from CCCP-induced mitochondrial fragmentation. (A–F) Cells were transfected with scrambled (A–C) or Yme1 siRNA oligonucleotides (D–F). Mitochondrial morphology was observed by cotransfection with mitochondrial targeted DsRed. Cells was observed without further treatment at 2 d after transfection (A and D), after incubation with CCCP for 60 min (B and E), or incubation with CCCP for 60 min followed by washout and 3.5-h recovery (C and F). (G) Percentages of cells with different mitochondrial morphologies and the treatments described in A–F. Morphologies were classified as connected (highly interconnected), normal (tubular morphology), intermediate (partially fragmented), and fragmented (fully fragmented). Samples sizes for the scrambled controls were as follows: n = 506 cells counted before the addition of CCCP, n = 246 counted after adding CCCP, and n = 100 cells counted at 3.5 h after washout. Sample sizes for Yme1 siRNA were n = 384, n = 332, and n = 169, respectively. Bar, 10 μm.
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