View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Mitochondrial Rhomboid PARL Regulates Cytochrome c Release during Apoptosis via OPA1-Dependent Cristae Remodeling Sara Cipolat,1,7 Tomasz Rudka,2,7 Dieter Hartmann,2,7 Veronica Costa,1 Lutgarde Serneels,2 Katleen Craessaerts,2 Kristine Metzger,2 Christian Frezza,1 Wim Annaert,3 Luciano D’Adamio,5 Carmen Derks,2 Tim Dejaegere,2 Luca Pellegrini,6 Rudi D’Hooge,4 Luca Scorrano,1,* and Bart De Strooper2,* 1 Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, Padova, Italy 2 Neuronal Cell Biology and Gene Transfer Laboratory 3 Membrane Trafficking Laboratory Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology (VIB4) and K.U.Leuven, Leuven, Belgium 4 Laboratory of Biological Psychology, K.U.Leuven, Leuven, Belgium 5 Albert Einstein College of Medicine, Bronx, NY 10461, USA 6 Centre de Recherche Robert Giffard, Universite` Laval, G1J 2G3 Quebec, Canada 7 These authors contribute equally to this work. *Contact: [email protected] (L.S.); [email protected] (B.D.S.) DOI 10.1016/j.cell.2006.06.021 SUMMARY been identified in D. melanogaster (Freeman, 2004), where they function as essential activators of the epidermal Rhomboids, evolutionarily conserved integral growth factor (EGF) signaling pathway, proteolytically membrane proteases, participate in crucial sig- cleaving the EGF receptor ligands Spitz, Gurken, and naling pathways. Presenilin-associated rhom- Keren. Since all Rhomboids share a conserved serine boid-like (PARL) is an inner mitochondrial protease catalytic dyad (Lemberg et al., 2005), it has membrane rhomboid of unknown function, been suggested that they are able to cleave proteins in whose yeast ortholog is involved in mito- the transmembrane domain. Therefore, together with the presenilin aspartyl proteases and the Site 2 metallo- chondrial fusion. ParlÀ/À mice display normal proteases, they have been functionally assigned to a intrauterine development but from the fourth previously unidentified class of highly hydrophobic prote- postnatal week undergo progressive multi- ases involved in ‘‘regulated intramembranous proteolytic systemic atrophy leading to cachectic death. cleavage,’’ a novel cell-signaling mechanism (Brown Atrophy is sustained by increased apoptosis, et al., 2000). Our knowledge of the mammalian rhomboids both in and ex vivo. ParlÀ/À cells display normal is extremely scarce. For example, they are unlikely to be mitochondrial morphology and function but are involved in EGF signaling, since TGFa, the major mam- no longer protected against intrinsic apoptotic malian ligand of the EGFR pathway, is released by metal- death stimuli by the dynamin-related mitochon- loproteases of the ADAM family (Freeman, 2004). drial protein OPA1. ParlÀ/À mitochondria dis- Recently, a mitochondrial rhomboid, rbd1/pcp1, was play reduced levels of a soluble, intermembrane identified in Saccharomyces cerevisiae (Esser et al., 2002; Herlan et al., 2003; McQuibban et al., 2003; Sesaki space (IMS) form of OPA1, and OPA1 spe- À/À et al., 2003). Drbd1 cells display fragmented mitochondria cifically targeted to IMS complements Parl and impaired growth on nonfermentable carbon sources, cells, substantiating the importance of PARL similar to the phenotype caused by deletion of the dyna- À/À in OPA1 processing. Parl mitochondria un- min-related protein Mgm1p, which turned out to be a sub- dergo faster apoptotic cristae remodeling and strate for Rbd1p. The short isoform of Mgm1p produced cytochrome c release. These findings implicate by Rbd1p is required to maintain mitochondrial morphol- regulated intramembrane proteolysis in con- ogy and fusion (Herlan et al., 2003; McQuibban et al., trolling apoptosis. 2003). Thus, rhomboids and intramembrane proteolysis appear to control mitochondrial dynamics and function in yeast. INTRODUCTION Mitochondria are crucial organelles in intermediate metabolism and energy production (Danial et al., 2003), Rhomboid proteases constitute probably the most widely Ca2+ signaling (Rizzuto et al., 2000), and integration and conserved polytopic-membrane-protein family identified amplification of apoptotic signals (Green and Kroemer, until now (Koonin et al., 2003). Seven rhomboids have 2004). Such functional versatility is mirrored by their Cell 126, 163–175, July 14, 2006 ª2006 Elsevier Inc. 163 complex and dynamic morphology, controlled by a grow- ing family of ‘‘mitochondria-shaping’’ proteins that regu- late fusion and fission events. In mammals, fission is con- trolled by the dynamin-related protein DRP-1 (Smirnova et al., 2001) and its outer membrane (OM) adaptor hFis1 (James et al., 2003; Yoon et al., 2003). Fusion is mediated by two OM proteins, mitofusin (MFN) -1 and -2. Optic atro- phia 1 (OPA1), the homolog of S. cerevisiae Mgm1p, is the only dynamin-related protein identified in the inner membrane (IM) so far (Olichon et al., 2002). OPA1 pro- motes mitochondrial fusion by cooperating with MFN1 (Cipolat et al., 2004) and is mutated in dominant optic atrophy, the most common cause of inherited optic neu- ropathy (Alexander et al., 2000; Delettre et al., 2000). The homolog of yeast Rbd1p in mammals is the so-called ‘‘presenilin-associated rhomboid-like’’ (PARL). PARL is a rhomboid protease originally identified in a two-hybrid screen to interact with presenilin, the enzymatically active core protein of g-secretase (Pellegrini et al., 2001), and later found to be mitochondrial (Sik et al., 2004). We used a genetic approach to investigate the function of PARL and its potential role in OPA1 processing. RESULTS Targeted Inactivation of the Mouse Parl Gene Mice with loxP sites inserted in the Parl gene (Parlflx/flx) were generated by homologous recombination (Fig- ure 1A). They were crossed with a mouse strain express- ing Cre from the PGK promoter, resulting in Cre-mediated excision of the region between the loxP sites in all tissues. The resulting Parl null allele (ParlÀ/À)(Figure 1A) still gener- ated a small amount of aberrantly migrating RNA (Fig- ure 1C). RT-PCR and sequencing showed the absence of exon 2. Since this resulted in a frame shift and prema- ture stop codon, the remaining RNA is no longer functional Figure 1. Generation of Parl Knockout Mice (Figures 1D and 1F). Immunoblotting confirmed the loss of (A) Maps of the targeting vector, the wild-type Parl allele, the condi- PARL in fibroblasts derived from ParlÀ/À mice (Figure 1E). tional targeted allele (floxed allele), and the disrupted Parl allele. Exons (black boxes), LoxP and FRT recombination sites (white and black ParlÀ/À Mice Prematurely Die of Progressive arrowheads, respectively), and locations of PCR primers are indicated. The expected sizes for restriction fragments detected by 50 (L), 30 (R) Cachexia flanking or internal probes (H) (PCR fragments, black bars) from À/À Parl mice were born in a normal Mendelian frequency targeted and wild-type alleles are indicated with line diagrams. Posi- and developed normally up to 4 weeks. From then on, tive selection marker is indicated as a gray box. Relevant restriction mice displayed severe growth retardation (Figures 2A sites are shown (BglII, indicated as B and EcoRI as E). and 2B). ParlÀ/À mice lost muscle mass (erector spinae, (B) Examples of Southern blot of DNA isolated from ES cells, digested abdominal muscles, and diaphragm), leading to postural with EcoRI, and hybridized with the different probes (L, R, and internal [H]). defects with hunchback deformity (Figure 2A). All animals (C) Northern blot: the Parl transcript is detected in wt and heterozygous died between 8 and 12 weeks (Figure 2C), most likely as embryos. In ParlÀ/À, a weak signal corresponding to an aberrant a consequence of moving and breathing problems and transcript is detected. The b-actin transcript is detected as control. general cachexia. (D) RT-PCR analysis of Parl transcripts in wt and ParlÀ/À MEF cells. Microscopically, the diameter of individual muscle A shorter transcript of Parl is detected in the ParlÀ/À cells. Sequencing fibers was reduced (Figure 2O). Spinal motoneurons of the aberrant transcript confirmed a reading shift in remaining tran- script. were normal, and AChE histochemistry failed to reveal (E) 100 mg of wt and ParlÀ/À MEFs lysate was resolved by SDS-PAGE signs of neurogenic atrophy (data not shown). At 8 weeks, and probed with anti-PARL antibody (specific band around 30 kDa). thymus and spleen were massively atrophic, weighing The unspecific upper band was used as loading control. 10% or less compared to controls (Figures 2D and 2I), (F) Prediction of the maximal possible aberrant PARL protein. Bold with severe lymphocyte loss (Figures 2E and 2J). Uteri re- amino acids are identical to wt PARL. mained prepuberal, while ovaries were histologically 164 Cell 126, 163–175, July 14, 2006 ª2006 Elsevier Inc. normal. Males showed cryptorchidism with size reduction unchanged, while DP cells were reduced over 100-fold of testes, epidydimis, and accessory glands (data not in ParlÀ/À mice (Figures 2F and 2G) as a result of massive shown). Fluoro-Jade (Figure 2N) staining and activated apoptosis revealed by their counterstaining with annexin- caspase-3 immunoreactivity (data not shown) indicated V(Figure 2H, gray bars). Similarly, apoptosis depleted the (mild) neurodegenerative changes and apoptotic cell B220+ B cell population in ParlÀ/À spleens (Figures 2K– death in thalamus and striatum. Latency of the acoustic 2M). ParlÀ/À thymocytes isolated