The yeast protein cytochrome c peroxidase (Ccp1) is nuclearly encoded and imported into the mitochondrial intermembrane space, where it is involved in degradation of reactive oxygen species. It is known, that Ccp1 is synthesised as a precursor with a N-terminal pre-sequence, that is proteolytically removed during transport of the protein. This study presents evidence for a new processing pathway, involving novel signal peptidase activities. The mAAA protease subunits Yta10 (Afg3) and Yta12 (Rca1) were identified both to be essential for the first processing step. In addition, the Pcp1 (Ygr101w) gene product was found to be required for the second processing step, yielding the mature Ccp1 protein. The newly identified Pcp1 protein belongs to the rhomboid-GlpG superfamily of putative intramembrane peptidases. Inactivation of the protease motifs in mAAA and Pcp1 blocks the respective steps of proteolysis. A model of coupled Ccp1 transport and N-terminal processing by the mAAA complex and Pcp1 is discussed. Similar processing mechanisms may exist, because the mAAA subunits and the newly identified Pcp1 protein belong to ubiquitous protein families (Esser, 2002).
The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, associated with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation. Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, these isoforms have been characterized. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphology. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Deltapcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Deltamgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphology and maintenance of mitochondrial DNA (Herlan, 2003).
The dynamin-related GTPase, Mgm1p, is critical for the fusion of the mitochondrial outer membrane, maintenance of mitochondrial DNA (mtDNA), formation of normal inner membrane structures, and inheritance of mitochondria. Although there are two forms of Mgm1p, 100 and 90 kDa, their respective functions and the mechanism by which these two forms are produced are not clear. ugo2 mutants were isolated in a genetic screen to identify components involved in mitochondrial fusion. ugo2 mutants are defective in PCP1, a gene encoding a rhomboid-related serine protease. Cells lacking Pcp1p are defective in the processing of Mgm1p and produce only the larger (100 kDa) form of Mgm1p. Similar to mgm1delta cells, pcp1delta cells contain partially fragmented mitochondria, instead of the long tubular branched mitochondria of wild-type cells. In addition, pcp1delta cells, like mgm1delta cells, lack mtDNA and therefore are unable to grow on nonfermentable medium. Mutations in the catalytic domain lead to complete loss of Pcp1p function. Similar to mgm1delta cells, the fragmentation of mitochondria and loss of mtDNA of pcp1delta cells were rescued when mitochondrial division was blocked by inactivating Dnm1p, a dynamin-related GTPase. Surprisingly, in contrast to mgm1delta cells, which are completely defective in mitochondrial fusion, pcp1delta cells can fuse their mitochondria after yeast cell mating. This study demonstrates that Pcp1p is required for the processing of Mgm1p and controls normal mitochondrial shape and mtDNA maintenance by producing the 90 kDa form of Mgm1p. However, the processing of Mgm1p is not strictly required for mitochondrial fusion, indicating that the 100 kDa form is sufficient to promote fusion (Sesaki, 2003b).
Mitochondrial morphology and inheritance of mitochondrial DNA in yeast depend on the dynamin-like GTPase Mgm1. It is present in two isoforms in the intermembrane space of mitochondria both of which are required for Mgm1 function. Limited proteolysis of the large isoform by the mitochondrial rhomboid protease Pcp1/Rbd1 generates the short isoform of Mgm1 but how this is regulated is unclear. Near its NH2 terminus Mgm1 contains two conserved hydrophobic segments of which the more COOH-terminal one is cleaved by Pcp1. Changing the hydrophobicity of the NH2-terminal segment modulated the ratio of the isoforms and led to fragmentation of mitochondria. Formation of the short isoform of Mgm1 and mitochondrial morphology further depend on a functional protein import motor and on the ATP level in the matrix. These data show that a novel pathway, to which is referred to as alternative topogenesis, represents a key regulatory mechanism ensuring the balanced formation of both Mgm1 isoforms. Through this process the mitochondrial ATP level might control mitochondrial morphology (Herlan, 2004).
Maturation of cytochrome c peroxidase (Ccp1) in mitochondria occurs by the subsequent action of two conserved proteases in the inner membrane: the m-AAA protease, an ATP-dependent protease degrading misfolded proteins and mediating protein processing, and the rhomboid protease Pcp1, an intramembrane cleaving peptidase. Neither the determinants preventing complete proteolysis of certain substrates by the m-AAA protease, nor the obligatory requirement of the m-AAA protease for rhomboid cleavage is currently understood. This study describes an intimate and unexpected functional interplay of both proteases. The m-AAA protease mediates the ATP-dependent membrane dislocation of Ccp1 independent of its proteolytic activity. It thereby ensures the correct positioning of Ccp1 within the membrane bilayer allowing intramembrane cleavage by rhomboid. Decreasing the hydrophobicity of the Ccp1 transmembrane segment facilitates its dislocation from the membrane and renders rhomboid cleavage m-AAA protease-independent. These findings reveal for the first time a non-proteolytic function of the m-AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid (Tatsuta, 2007).
Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signalling in Drosophila. Rhomboids are conserved throughout evolution, and even in eukaryotes their existence in species with no EGFRs implies that they must have additional roles. Saccharomyces cerevisiae has two rhomboids, which have been 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. Two substrates of Rbd1p have been identified: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion. Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Delta mitochondrial phenotype. The 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, PARL (Pellegrini, 2001), rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases (McQuibban, 2003).
Despite their widespread conservation, the only known function of eukaryotic rhomboid proteases is the activation of EGFR signalling in Drosophila. Their function was therefore examined in S. cerevisiae, which has no receptor tyrosine kinases but has two genes encoding rhomboids. Deletion of RBD1 (Deltarbd1) caused cells to grow slowly, whereas deletion of RBD2 had no obvious phenotype and did not enhance the phenotype of rbd1Delta cells. The rbd1Delta phenotype was rescued by plasmid-borne expression of RBD1, confirming that the slow growth was indeed caused by loss of RBD1 (McQuibban, 2003).
Although previously unnoticed, the Rbd1p sequence contains a signature motif for mitochondrial localization, as predicted by MitoProt and other algorithms. To test this prediction, homologous recombination was used to replace the wild-type gene with a fully functional gene fused at its carboxy terminus to green fluorescent protein (GFP). Rbd1p−GFP colocalized precisely with antibodies against yeast porin, a mitochondrial protein, showing that Rbd1p is restricted to the mitochondria (McQuibban, 2003).
Rbd1p is an integral membrane protein with six predicted transmembrane domains (TMDs), and it was localized to the mitochondrial inner membrane by monitoring the protease digestion of intact mitochondria. 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 membrane. Combined with the slow growth phenotype, the mitochondrial location of Rbd1p suggested that rbd1Delta cells might have a mitochondrial defect. Consistent with this, the rbd1Delta strain fails to grow on the non-fermentable carbon source glycerol, suggesting that it is deficient in respiration (McQuibban, 2003).
To test directly the possibility that loss of Rbd1p causes mitochondrial defects, mutant cells were examined by electron microscopy, and the cells lacked wild-type mitochondria. The cells appeared otherwise normal, and other intracellular structures were indistinguishable from wild-type controls. A mitochondrion-specific dye was used 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 rbd1Delta cells appear as small fragments and aggregated masses throughout the cell. In further support of mitochondrial defects, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining detected a loss of nucleoid structures, which represent mitochondrial genomes (McQuibban, 2003).
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, whether wild-type mitochondrial morphology and growth rates depended on the catalytic activity of Rbd1p was tested. Expression of wild-type RBD1 rescued the rbd1Delta cells, but a mutant form of the gene, in which the catalytic serine is replaced by glycine, failed to rescue the phenotype. This indicates that the intramembrane proteolytic activity of Rbd1p is required for its mitochondrial function (McQuibban, 2003).
To investigate further this requirement for a mitochondrial protease, potential Rbd1p substrates were identified 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 peroxidase; Mcr1p, an NADH-cytochrome b5 reductase; Mgm1p, a dynamin-like GTPase; Osm1p, a fumarate reductase; and Pet100p, a chaperone required by cytochrome c oxidase. Deletion strains of each of these candidate substrates were examined in four assays of mitochondrial function: overall growth rate, peroxide sensitivity, growth on glycerol and mitochondrial morphology. Only mgm1Delta was indistinguishable in behaviour from rbd1Delta (McQuibban, 2003).
Whether any of the candidate substrates undergo Rbd1p-dependent processing in vivo was examined 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 rbd1Delta strains. Whereas Mcr1p, Osm1p and Pet100p were unaffected by the loss of Rbd1p, Ccp1p and Mgm1p were cleaved in an Rbd1p-dependent way. In late log phase, all of Ccp1p and about 50% of Mgm1p were processed in wild-type cells. Both proteins, however, were uncleaved in rbd1Delta cells (McQuibban, 2003).
Proteolytic cleavage was rescued in the rbd1Delta strain by a wild-type copy of RBD1 but not by a catalytically inactive mutant form, 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 rbd1Delta cells (which depends on a different mitochondrial protease) indicates that Rbd1p is not generally required for cleavage of mitochondrial proteins. These data explain the identical phenotype of mgm1Delta and rbd1Delta and, coupled with the very weak phenotype of ccp1Delta, strongly suggest that Mgm1p is the primary substrate of the proteolytic function of Rbd1p in maintaining the integrity of the mitochondrial membrane. These results are also consistent with data implying that Mgm1p regulates mitochondrial membrane fusion (McQuibban, 2003).
The 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, the amounts of Rbd1p protein and cleavage of Mgm1p during were examined 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%. 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 (McQuibban, 2003).
Analysis of Mgm1p sequences from divergent yeasts 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 substrate. To test this prediction, amino acids 101−103 of Mgm1p (GGM to VVL) were altered and the ability of this TMD mutation to rescue the mgm1Delta strain was tested. This mutant did not complement the mitochondrial morphology or growth of the mgm1Delta strain and was uncleaved in cell extracts. These data support the hypothesis that Rbd1p cleaves Mgm1p in this proposed TMD (McQuibban, 2003).
The rhomboid protease family is widely conserved and, using the MitoProt algorithm for prediction of mitochondrial targeting domains, it was found that mitochondrial rhomboids occur throughout the eukaryotes: in Drosophila, it is Rhomboid-7; and in mammals, PARL (presenilin-associated rhomboid-like. 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. The possibility was examined that the function, as well as the location, of mitochondrial rhomboids is conserved, by testing whether human PARL could rescue the rbd1Delta mutation. The expression of PARL in rbd1Delta cells restored Ccp1p and Mgm1p processing and also rescued growth rate and mitochondrial morphology. This suggests that the mitochondrial function of Rbd1p might be conserved in mammals. The 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 (McQuibban, 2003).
Mitochondria are dynamic organelles, frequently undergoing marked remodelling. 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 dynamics. Mgm1p functions in the intermembrane space and regulates membrane fusion by interacting with the outer membrane proteins Fzo1p and Ugo1p. The current data imply that Mgm1p is initially an integral inner membrane protein; consistent with this, full-length and cleaved forms of Mgm1p show differential membrane association. The 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, is the cause of the most common form of childhood-onset blindness, dominant optic atrophy. It also has a predicted TMD and regulates mitochondrial membrane dynamics. Because human PARL can complement rbd1Delta, it is an intriguing possibility that the human mitochondrial rhomboid might also be involved in regulating the activity of OPA1 (McQuibban, 2003).
The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2 (PS1 and PS2), are involved in amyloid beta-protein precursor processing (AbetaPP), Notch receptor signaling, and programmed cell death. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. Clues about the function of a protein can be obtained by seeing whether it interacts with another protein of known function. Using the yeast two-hybrid system, two proteins were identifed that interact and colocalize with the presenilins. One of these newly detected presenilin-interacting proteins belongs to the FtsH family of ATP-dependent proteases, and the other one belongs to Rhomboid superfamily of membrane proteins that are highly conserved in eukaryotes, archaea and bacteria. Based on the pattern of amino acid residues conservation in the Rhomboid superfamily, it is hypothesized that these proteins possess a metal-dependent enzymatic, possibly protease activity. The two putative proteases interacting with presenilins could mediate specific proteolysis of membrane proteins and contribute to the network of interactions in which presenilins are involved (Pellegrini, 2001).
Regulated intramembrane proteolysis (RIP) is an emerging paradigm in signal transduction. RIP is mediated by intramembrane-cleaving proteases (I-CliPs), which liberate biologically active nuclear or secreted domains from their membrane-tethered precursor proteins. The yeast Pcp1p/Rbd1p protein is a Rhomboid-like I-CliP that regulates mitochondrial membrane remodeling and fusion through cleavage of Mgm1p, a regulator of these essential activities. Although this ancient function is conserved in PARL (Presenilins-associated Rhomboid-like protein), the mammalian ortholog of Pcp1p/Rbd1p, the two proteins show a strong divergence at their N termini. However, the N terminus of PARL is significantly conserved among vertebrates, particularly among mammals, suggesting that this domain evolved a distinct but still unknown function. This study shows that the cytosolic N-terminal domain of PARL is cleaved at positions 52-53 (alpha-site) and 77-78 (beta-site). Whereas alpha-cleavage is constitutive and removes the mitochondrial targeting sequence, beta-cleavage appears to be developmentally controlled and dependent on PARL I-CliP activity supplied in trans. The beta-cleavage of PARL liberates Pbeta, a nuclear targeted peptide whose sequence is conserved only in mammals. Thus, in addition to its evolutionarily conserved function in regulating mitochondrial dynamics, PARL might mediate a mammalian-specific, developmentally regulated mitochondria-to-nuclei signaling through regulated proteolysis of its N terminus and release of the Pbeta peptide (Sik, 2004).
Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis (Cipolat, 2006).
Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. This study shows that phosphorylation and cleavage of the vertebrate-specific Pbeta domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position Ser(77)-Ala(78) that is required to initiate PARL-induced mitochondrial fragmentation. These findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology (Jeyaraju, 2006).
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