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. 2007 Sep;18(9):3582-90.
doi: 10.1091/mbc.e07-02-0164. Epub 2007 Jul 5.

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

Stéphane Duvezin-Caubet  1 , Mirko Koppen, Johannes Wagener, Michael Zick, Lars Israel, Andrea Bernacchia, Ravi Jagasia, Elena I Rugarli, Axel Imhof, Walter Neupert, Thomas Langer, Andreas S Reichert
Affiliations

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

Stéphane Duvezin-Caubet et al. Mol Biol Cell. 2007 Sep.

Abstract

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.

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Figures

Figure 1.
Figure 1.
Characterization of OPA1 isoforms by mass spectrometry. (A) Immunoprecipitation of OPA1 from isolated HeLa mitochondria (15 mg) by using anti-OPA1 antibodies. Equal fractions (0.25%) of total mitochondria (T), supernatant (S), and pellet (P) of solubilized mitochondria after clarifying spin, flow through (FT) and 1% of the elution fraction (E) were analyzed by SDS-PAGE and immunoblotting with anti-OPA1 antibodies. (B) Coomassie-stained bands of immunoprecipitated OPA1 isoforms. Each band was separately cut and used for ESI-LC-MS/MS. (C) Alignment of N termini of the eight OPA1 splice variants. MPP cleavage site N-terminal to F88 (Ishihara et al., 2006) is indicated. Vertical dotted lines indicate the exon boundaries. Gray highlighted areas represent hydrophobic stretches called TM1, TM2a, and TM2b (Herlan et al., 2004). Boxes represent peptides found by ESI-LC-MS/MS analysis of any of the different immunoprecipitated OPA1 isoforms. The absence (−) or presence (+) of each peptide in different OPA1 isoforms (L1–S5) is indicated below the alignment. The confidence of peptide identification is indicated by +++ (excellent), ++ (very good), or + (good) based on the number of identifications in separate ESI-LC-MS/MS runs and on the ion score. (D) OPA1 splice variant 7 (Sp.7) was expressed in wild-type yeast cells (left) or was overexpressed in HeLa cells (right), and total cell extracts thereof or mitochondria isolated from HeLa cells (M) were analyzed by Western blotting with anti-OPA1 antibodies. Endogenous OPA1 isoforms (endo) in HeLa cell extracts are shown for comparison. Bands are labeled according to apparent corresponding size of OPA1 isoforms in HeLa mitochondria (L1, L2, S3, S4, and S5). Precursor protein (p) and a degradation product (d) are indicated.
Figure 2.
Figure 2.
Processing of OPA1 in yeast does not depend on rhomboid proteases PARL or Pcp1. (A–D) Functional complementation of Pcp1 by the human mitochondrial rhomboid protease PARL. WT or Δpcp1 (Δ) spores expressing either the human (PARL) or the yeast (PCP1) mitochondrial rhomboid protease were used. (A) Total cell extracts of the indicated strains expressing OPA1 Sp.4, 7, or 8 were analyzed by Western blotting. For splice variant 8, one band was slightly larger than L1 from HeLa mitochondria (data not shown) and was therefore labeled L1′. This is consistent with the larger predicted size of the MPP-cleaved splice variant 8 compared with splice variant 7 forming L1. (B) Processing of the two known substrates of Pcp1 was analyzed by Western blotting of cell extracts of indicated strains. The bands indicated are the large isoform of Mgm1 (l-Mgm1), the small isoform of Mgm1 (s-Mgm1), a degradation fragment of Mgm1 (f; only in Δpcp1), the mature form of Ccp1 (mCcp1), and the intermediate form of Ccp1 (iCcp1; only in Δpcp1). For control, anti-Pcp1 immunodecoration is shown. (C and D) Analysis of mitochondrial morphology of the indicated strains expressing a mitochondrially targeted GFP. (C) Representative images (top, bright field; bottom, fluorescence). (D) Quantification of mitochondrial morphology. Error bars represent the SD (n = 3). (E) Cultured mouse embryonic fibroblasts isolated from Parl+/+ (WT) and Parl−/− mice were treated or not with 20 μM CCCP for 30 min. Cell extracts were subjected to Western blotting with anti-OPA1 antibodies. HeLa mitochondria (M) were used as a control.
Figure 3.
Figure 3.
OPA1 processing depends on the m-AAA protease. (A) OPA1 splice variant 7 was expressed in yeast strains bearing deletions of putative or known mitochondrial proteases and in WT and analyzed by Western blotting. HeLa mitochondria (M) are shown for comparison. (B) Wild-type and Δyta10Δyta12 cells complemented (+) or not (−) with their human orthologues AFG3L2 and paraplegin (L2/PARA) expressing OPA1 Sp.4, 7, or 8. Total cell extracts and HeLa mitochondria (M) were analyzed by Western blotting. (C) OPA1 splice variants 4, 7, and 8 were expressed in Δyta10Δyta12 with (+) and without (−) PARL. Cell lysates were analyzed by Western blotting. (D) Cultured mouse fibroblasts isolated from Spg7+/+ (WT) and Spg7−/− mice were treated or not with 20 μM CCCP. Total cell extracts were subjected to Western blotting.
Figure 4.
Figure 4.
OPA1 processing by homo-oligomeric human m-AAA protease complexes in the absence of paraplegin. Human OPA1 Sp.4, 7, or 8 were expressed in WT and in Δyta10Δyta12 cells harboring the human AFG3L2 or the proteolytically inactive variant AFG3L2E575Q as indicated. Total cell extracts were analyzed by Western blotting. HeLa total cell extract was used as reference.
Figure 5.
Figure 5.
OPA1 processing depends on the subunit composition of the murine m-AAA protease. OPA1 was expressed in WT; Δyta10Δyta12 cells; or Δyta10Δyta12 cells harboring either murine paraplegin (para), Afg3l1, Afg3l2, or their mutant variants paraplegin E575Q (paraEQ), Afg3l1E567Q (Afg3l1EQ), or Afg3l2E574Q (Afg3l2EQ) or combinations of them.
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