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Mitochondrial membrane remodelling regulated by a conserved rhomboid protease

Abstract

Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signalling in Drosophila1. Rhomboids are conserved throughout evolution2,3,4,5, and even in eukaryotes their existence in species with no EGFRs implies that they must have additional roles. Here we report that Saccharomyces cerevisiae has two rhomboids, which we have named Rbd1p and Rbd2p. RBD1 deletion results in a respiratory defect; consistent with this, Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria. We have identified two substrates of Rbd1p: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion6,7,8,9,10. Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Δ mitochondrial phenotype. Our data indicate that mitochondrial membrane remodelling is regulated by cleavage of Mgm1p and show that intramembrane proteolysis by rhomboids controls cellular processes other than signalling. In addition, mitochondrial rhomboids are conserved throughout eukaryotes and the mammalian homologue, PARL11, rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases.

Main

Despite their widespread conservation, the only known function of eukaryotic rhomboid proteases is the activation of EGFR signalling in Drosophila12,13,14,15. We therefore examined their function in S. cerevisiae, which has no receptor tyrosine kinases but has two genes encoding rhomboids, which we have named RBD1 (YGR101w) and RBD2 (YPL246c). Deletion of RBD1 (Δrbd1) caused cells to grow slowly (Fig. 1a), whereas deletion of RBD2 had no obvious phenotype and did not enhance the phenotype of rbd1Δ cells (data not shown). The rbd1Δ phenotype was rescued by plasmid-borne expression of RBD1 (Fig. 1a), confirming that the slow growth was indeed caused by loss of RBD1.

Figure 1: Characterization of Rbd1p.
figure 1

a, rbd1Δ cells grow more slowly than wild type. This growth defect is rescued by plasmid-borne expression of RBD1. b, Optical sections through yeast spheroplasts indicate the mitochondrial localization of Rbd1p–GFP (red), as shown by its colocalization with yeast mitochondrial porin (green). c, Rbd1p has an inner mitochondrial membrane location. Enriched mitochondrial fractions were treated with increasing amounts of proteinase K (lanes 1–4, 0, 10, 50 and 100 µg ml-1; lanes 5, 100 µg ml-1 plus 0.1% Triton X-100). Rbd1p was protected from degradation except in the presence of detergent. Compare the markers F1β and Tom40p, which are respectively inner and outer membrane proteins. d, Electron microscopy shows a loss of normal mitochondria in rbd1Δ cells. The nucleus (n) and vacuole (v) are labelled, and normal mitochondria are indicated with black arrowheads. e, Vital staining of wild-type and rbd1Δ cells with Mitotracker Green FM. f, DAPI staining of wild-type and rbd1Δ cells, showing loss of nucleoids. g, Mitochondrial morphology defects are rescued by a plasmid-borne wild-type copy of RBD1 (91% wild-type mitochondria, 9% fragmented/aggregated, n = 100) but not mutant RBD1 (0% wild-type mitochondria, 100% fragmented/aggregated, n = 100). h, Growth of rbd1Δ is also fully restored by wild-type but not mutant RBD1.

Although previously unnoticed, the Rbd1p sequence contains a signature motif for mitochondrial localization, as predicted by MitoProt16 and other algorithms. To test this prediction, we used homologous recombination to replace the wild-type gene with a fully functional gene fused at its carboxy terminus to green fluorescent protein (GFP; see Supplementary Fig. S1). Rbd1p–GFP colocalized precisely with antibodies against yeast porin, a mitochondrial protein, showing that Rbd1p is restricted to the mitochondria (Fig. 1b).

Rbd1p is an integral membrane protein with six predicted transmembrane domains (TMDs), and we localized it to the mitochondrial inner membrane by monitoring the protease digestion of intact mitochondria (Fig. 1c). This clearly distinguishes Rbd1p from previously analysed eukaryotic rhomboids, all of which have been found to be located in the secretory pathway or on the plasma membrane17. Combined with the slow growth phenotype, the mitochondrial location of Rbd1p suggested that rbd1Δ cells might have a mitochondrial defect. Consistent with this, the rbd1Δ strain failed to grow on the non-fermentable carbon source glycerol (Fig. 2b), suggesting that it was deficient in respiration.

Figure 2: Phenotypes of candidate Rbd1p substrates.
figure 2

a, Growth of five strains with deletions of potential substrates is compared with the growth of rbd1Δ. Only mgm1Δ and pet100Δ show growth defects similar to rbd1Δ. The relative sensitivity of each strain to H2O2 is shown on the right. rbd1Δ, ccp1Δ, mcr1Δ and mgm1Δ are sensitive to H2O2. b, Growth on glycerol-containing medium shows that rbd1Δ, mgm1Δ and pet100Δ are respiration-deficient. c, Of the candidates tested, the mitochondrial morphology (as detected by Mitotracker Green FM) of only mgm1Δ resembles that of rbd1Δ. In all tests, rbd2Δ was included as a control for specificity of the phenotypes.

To test directly the possibility that loss of Rbd1p caused mitochondrial defects, we examined mutant cells by electron microscopy and found that they lacked wild-type mitochondria (Fig. 1d). The cells appeared otherwise normal, and other intracellular structures were indistinguishable from wild-type controls. We next used a mitochondrion-specific dye to examine living cells. Mitochondria from wild-type yeast cells in log-phase growth generally appear as tubular structures around the cell cortex. By contrast, the mitochondria of rbd1Δ cells appeared as small fragments and aggregated masses throughout the cell (Fig. 1e). In further support of mitochondrial defects, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining detected a loss of nucleoid structures, which represent mitochondrial genomes (Fig. 1f).

The requirement for a rhomboid in the maintenance of mitochondrial morphology and genome maintenance was unexpected and suggested two possibilities. Either intramembrane proteolytic activity similar to that which activates intercellular signalling in other cases is used in a different context, or some other uncharacterized feature of the rhomboid protein is responsible for its mitochondrial function. To distinguish between these possibilities, we tested whether wild-type mitochondrial morphology and growth rates depended on the catalytic activity of Rbd1p. Expression of wild-type RBD1 rescued the rbd1Δ cells, but a mutant form of the gene, in which the catalytic serine is replaced by glycine1, failed to rescue the phenotype (Fig. 1g, h). This indicates that the intramembrane proteolytic activity of Rbd1p is required for its mitochondrial function.

To investigate further this requirement for a mitochondrial protease, we identified potential Rbd1p substrates by selecting proteins from the yeast genome that met the following criteria: a mitochondrial localization, the presence of a single predicted TMD and experimental evidence that the protein is soluble, which together suggest that the protein may undergo proteolytic cleavage. Five characterized proteins fulfilled these criteria: Ccp1p, a cytochrome c peroxidase18; Mcr1p, an NADH-cytochrome b5 reductase19; Mgm1p, a dynamin-like GTPase6; Osm1p, a fumarate reductase20; and Pet100p, a chaperone required by cytochrome c oxidase21. We examined deletion strains of each of these candidate substrates in four assays of mitochondrial function: overall growth rate, peroxide sensitivity, growth on glycerol and mitochondrial morphology (Fig. 2a–c). Only mgm1Δ was indistinguishable in behaviour from rbd1Δ.

We examined directly whether any of the candidate substrates undergo Rbd1p-dependent processing in vivo, by replacing the wild-type genome copy of the gene with a C-terminal haemagglutinin A (HA)-tagged copy, both in the wild-type and rbd1Δ strains. Whereas Mcr1p, Osm1p and Pet100p were unaffected by the loss of Rbd1p, Ccp1p and Mgm1p were cleaved in an Rbd1p-dependent way (Fig. 3a). In late log phase, all of Ccp1p and about 50% of Mgm1p were processed in wild-type cells. Both proteins, however, were uncleaved in rbd1Δ cells; this requirement for Rbd1p in Ccp1p processing confirms the results of another study22.

Figure 3: Cleavage of Ccp1p and Mgm1p depends on Rbd1p in vivo.
figure 3

a, Western blot analysis of cell lysates from cultures at late log phase shows that Ccp1p and Mgm1p are each processed to a faster-migrating species (arrowheads) in wild type but not in rbd1Δ. By contrast, Mcr1p, Osm1p and Pet100p are unaffected by the loss of Rbd1p. b, Cleavage of both Ccp1p and Mgm1p is restored in rbd1Δ strains by plasmid-borne expression of RBD1 (lanes 3) but not a catalytic mutant form of RBD1 (lanes 4). Plasmid-borne expression of the human homologue of RBD1 (hPARL) also restores cleavage of both Ccp1p and Mgm1p (lanes 5). Lanes 1, cell lysates from wild type; lanes 2, cell lysates from rbd1Δ. c, Western blot analysis of Mgm1p and Rbd1p (using antibodies against HA and GFP, respectively) in cell lysates from wild-type cells as they exit logarithmic growth. Time points from left to right are 0, 2, 4, 6, 8, 10 and 12 h, starting at an absorbance at 600 nm of 0.2. Growth curves indicate that cells start to exit logarithmic phase at 6 h (arrowheads). d, Alignment of the N termini of Mgm1p from S. cerevisiae and from two other yeast species, Saccharomyces castelli and Saccharomyces kluyveri (P. Cliften and M. Johnston, personal communication). Horizontal line indicates the predicted TMD; VVL indicates the position of the TMD mutant used in e. e, Mitochondrial morphology defects are rescued by plasmid-borne wild-type copy of MGM1 (83% wild-type mitochondria, 17% fragmented/aggregated, n = 100) but not a mutant copy of MGM1, in which TMD residues 101–103 have been replaced with VVL (0% wild-type mitochondria, 100% fragmented/aggregated, n = 100). Western blot of lysates of cultures in logarithmic phase shows that the VVL mutant Mgm1p is not efficiently cleaved. f, The slow growth phenotype is rescued by a plasmid-borne copy of wild-type MGM1 but not a copy of the VVL MGM1mutant.

Proteolytic cleavage was rescued in the rbd1Δ strain by a wild-type copy of RBD1 but not by a catalytically inactive mutant form (Fig. 3b), showing that, as with the mitochondrial phenotype, the processing of both Ccp1p and Mgm1p requires the intramembrane serine protease activity of Rbd1p. The normal processing of Mcr1p in rbd1Δ cells (which depends on a different mitochondrial protease19) indicates that Rbd1p is not generally required for cleavage of mitochondrial proteins. These data explain the identical phenotype of mgm1Δ and rbd1Δ and, coupled with the very weak phenotype of ccp1Δ, strongly suggest that Mgm1p is the primary substrate of the proteolytic function of Rbd1p in maintaining the integrity of the mitochondrial membrane. Our results are also consistent with data implying that Mgm1p regulates mitochondrial membrane fusion7,8,10.

Our data indicate that, in order to function in membrane fusion, Mgm1p needs to be activated by Rbd1p-catalysed intramembrane cleavage. Because mitochondrial morphology and requirements change significantly as cells move from exponential growth to stationary phase, we examined the amounts of Rbd1p protein and cleavage of Mgm1p during this transition period. As cells left logarithmic-phase growth, Rbd1p was downregulated and there was a simultaneous reduction of Mgm1p cleavage, from 95% to about 50% (Fig. 3c). Notably, the overall level of Mgm1p did not decrease over the time course of the experiment, implying that the reduction of Rbd1p did not simply reflect a general loss of mitochondrial protein. This suggests that expression of Rbd1p is a physiologically significant regulator of mitochondrial remodelling.

Analysis of Mgm1p sequences from divergent yeasts (Fig. 3d) identifies a highly conserved region that is predicted to form a TMD and represents a potential cleavage site for Rbd1p. Notably, this region has sequence characteristics (glycine and alanine residues) that suggest that it might be a rhomboid substrate23. To test this prediction, we altered amino acids 101–103 of Mgm1p (GGM to VVL) and tested the ability of this TMD mutation to rescue the mgm1Δ strain. This mutant did not complement the mitochondrial morphology or growth of the mgm1Δ strain and was uncleaved in cell extracts (Fig. 3e, f). These data support the hypothesis that Rbd1p cleaves Mgm1p in this proposed TMD.

The rhomboid protease family is widely conserved2,3,4,5 and, using the MitoProt algorithm for prediction of mitochondrial targeting domains, we found that mitochondrial rhomboids occur throughout the eukaryotes (Fig. 4a): in Drosophila, it is Rhomboid-7; and in mammals, PARL (presenilin-associated rhomboid-like; accession number AAG28519)11. Neither of these has an assigned function. The prediction of mitochondrial location was validated by expressing mouse PARL in COS cells, where it was localized exclusively to mitochondria (Fig. 4b). We also examined the possibility that the function, as well as the location, of mitochondrial rhomboids is conserved, by testing whether human PARL could rescue the rbd1Δ mutation. The expression of PARL in rbd1Δ cells restored Ccp1p and Mgm1p processing (Fig. 3b, lanes 5) and also rescued growth rate and mitochondrial morphology (data not shown). This suggests that the mitochondrial function of Rbd1p might be conserved in mammals. Our combined data identify a subclass of rhomboids that control mitochondrial membrane dynamics in yeast and provide evidence that their function and specificity is conserved in mammals.

Figure 4: Mitochondrial rhomboids are conserved in higher eukaryotes.
figure 4

a, Sequence alignment of the human (hPARL), mouse (mPARL) and Drosophila (Rhom7) mitochondrial rhomboids. Bars above the sequence indicate the predicted TMDs (using TMHMM v.2.0) and the three asterisks below the sequence indicate the putative catalytic triad residues1. b, Expression of mouse PARL in COS cells shows colocalization with the mitochondrion-specific dye Mitotracker.

Mitochondria are dynamic organelles, frequently undergoing marked remodelling24,25,26. A balance of fusion and fission events is thought to regulate this process and Mgm1p is one of a trio of dynamin-like GTPases, conserved from yeast to mammals, that control these membrane dynamics7,8. Mgm1p functions in the intermembrane space and regulates membrane fusion by interacting with the outer membrane proteins Fzo1p and Ugo1p10. Our data imply that Mgm1p is initially an integral inner membrane protein (Fig. 3d, e, and Supplementary Fig. S2); consistent with this, full-length and cleaved forms of Mgm1p show differential membrane association7. Our results also imply that intramembrane cleavage of the TMD of this GTPase may be an essential activation step for its function. This is a previously unrecognized mode of regulation for dynamin-like proteins and suggests that membrane tethering may be incompatible with dynamin-like membrane remodelling activity. Notably, heterozygosity for the mammalian homologue of Mgm1p, OPA1 (ref. 27), is the cause of the most common form of childhood-onset blindness, dominant optic atrophy28. It also has a predicted TMD and regulates mitochondrial membrane dynamics28. Because human PARL can complement rbd1Δ, it is an intriguing possibility that the human mitochondrial rhomboid might also be involved in regulating the activity of OPA1.

Methods

Construction of yeast strains

All genetic manipulation of yeast was done as described29. Disruptions of the S. cerevisiae open reading frames YGR101w (also called Pcp1p22) and YPL246c were made in the α-303 yeast strain. We tagged candidate substrates at the C terminus with the influenza HA sequence by homologous recombination. Integration was confirmed by polymerase chain reaction (PCR) analysis of mutant colonies. Mutant strains of candidate Rbd1p substrates and RBD1 and RBD2 were purchased from EUROSCARF. For rescue experiments, the RBD1 open reading frame plus 200 nucleotides at the 5′ and 3′ ends was amplified by PCR from yeast genomic DNA and cloned into the yeast vector pRS315 (ATCC); the MGM1 gene in pRS313 was a gift from M. Yaffe.

Mutant strains carrying test plasmids were created by either plasmid shuffle (RBD1 rescue) or by tetrad dissection of transformed diploids (RBD1 and MGM1 rescue). We carried out site-directed mutagenesis of the catalytic serine residue of RBD1 to glycine and the transmembrane region of MGM1 using the Quick-Change kit (Stratagene). The complementary DNA of human PARL (obtained from IMAGE consortium) was cloned into a derivative of pRS315 with the Pho5 promoter (a gift from J. Whyte). Antisera against Mgm1p were a gift from M. Yaffe. Antisera against Tom40p were a gift from B. Westermann. Antisera against F1β were a gift from R. Jensen.

Histology

Electron microscopy images were taken after staining yeast cells with permanganate and then staining sections with uranyl acetate. Live yeast cells were fluorescently labelled with Mitotracker Green FM according to the manufacturer's instructions (Molecular Probes). We carried out antibody staining on fixed spheroplasts from yeast cells, which were labelled with monoclonal antibodies against yeast porin (Molecular Probes) and a rabbit polyclonal antiserum generated against recombinant GFP12. Nucleoid bodies were stained with DAPI by standard protocols29.

Protease protection

Mitochondrion-enriched fractions were prepared as described30 from wild-type, RBD1:GFP and MGM1:HA yeast strains. We carried out protease protection assays as described8 and analysed samples by western blot using the indicated antibodies.

Phenotypic analysis

Growth was tested on plates containing 2% glucose. Respiration competence was tested by growth on plates containing 2% glycerol. We assessed peroxide sensitivity by measuring the zone of cell death around a 5-mm disk containing 5 µl of 30% hydrogen peroxide. All growth assays were done at 30 °C.

Cell culture

We cultured COS cells under standard conditions. The mouse PARL cDNA (a gift from B. De Strooper) was inserted into the expression vector pSGF5 (Stratagene) and was transfected using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen). Forty-eight hours after transfection, cells were stained with Mitotracker (Molecular Probes), fixed with 4% formaldehyde and counterstained with a monoclonal antibody against the C-terminal HA tag as described12.

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Acknowledgements

We thank H. Pelham, S. Munro and J. Whyte for help and support; B. De Strooper for discussions; members of our laboratory for advice; M. Yaffe, R. Jensen and B. Westermann for antibodies; and P. Cliften and M. Johnston for providing yeast sequences before publication. G.A.M. is supported by an EMBO long-term fellowship.

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McQuibban, G., Saurya, S. & Freeman, M. Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541 (2003). https://doi.org/10.1038/nature01633

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