InteractiveFly: GeneBrief
rhomboid-7: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
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Gene name - rhomboid-7
Synonyms - Cytological map position- 48D5-48D5 Function - protease Keywords - spermatogenesis, mitochondrial fusion, eye |
Symbol - rho-7
FlyBase ID: FBgn0033672 Genetic map position - 2R Classification - rhomboid family Cellular location - mitochondrial transmembrane |
In addition to being energy generators, mitochondria control many cellular processes including apoptosis. They are dynamic organelles, and the machinery of membrane fusion and fission is emerging as a key regulator of mitochondrial biology. A novel and conserved mitochondrial rhomboid intramembrane protease has been identified that controls membrane fusion in Saccharomyces cerevisiae by processing the dynamin-like GTPase, Mgm1, thereby releasing it from the membrane (McQuibban, 2003). The genetics of mitochondrial membrane dynamics has until now focused primarily on yeast. In Drosophila, the mitochondrial rhomboid (Rhomboid-7) is required for mitochondrial fusion during fly spermatogenesis and muscle maturation, both tissues with unusual mitochondrial dynamics. Mutations in Drosophila optic atrophy 1-like (Opa1-like or CG8479), the ortholog of yeast mgm1, display similar phenotypes, suggesting a shared role for Rhomboid-7 and Opa1-like, as with their yeast orthologs. Loss of human OPA1 leads to dominant optic atrophy, a mitochondrial disease leading to childhood onset blindness. rhomboid-7 mutant flies have severe neurological defects, evidenced by compromised signaling across the first visual synapse, as well as light-induced neurodegeneration of photoreceptors that resembles the human disease. rhomboid-7 mutant flies also have a greatly reduced lifespan (McQuibban, 2006).
Mitochondria form a tubular network in most eukaryotic cells, the result of a balanced set of opposing membrane fusion and fission reactions. Disturbance of this balance disrupts mitochondrial architecture and function. Mitochondria are surrounded by double membranes and must maintain effective separation from the surrounding cytoplasm; there is considerable complexity in the mechanism underlying the membrane dynamics required for fusion and fission. In Saccharomyces cerevisiae, two dynamin-related large GTPases, Mgm1 (Guan, 1993; Sesaki, 2003a) and Fzo1 (Rapaport, 1998; Hermann, 1998), regulate the fusion reaction, although their precise functions remain unknown. It has recently become clear that Mgm1 is regulated by a two-stage proteolytic release from the inner mitochondrial membrane: first, the mitochondrial targeting sequence is removed by MPP (mitochondrial processing peptidase), a matrix-localized protease; second, the intramembrane serine protease activity of the mitochondrial rhomboid family member Rbd1/Pcp1 releases a soluble form of Mgm1 into the intermembrane space (McQuibban, 2003; Herlan, 2004; Herlan, 2003; Sesaki, 2003a). Loss of Rbd1/Pcp1 leads to a phenotype similar to loss of Mgm1, implying that this intramembrane cleavage is necessary for full Mgm1 function (McQuibban, 2006).
Rbd1/Pcp1 and Mgm1 are conserved throughout eukaryotes, but much less is known about the genetic networks or mechanisms that control mitochondrial membrane dynamics in higher organisms. In humans, disruption of mitochondrial function is a major cause of inherited disease, including many neurological defects. Of particular relevance to thee studies, mutation of OPA1, the human ortholog of Mgm1, leads to autosomal dominant optic atrophy, the most common familial, childhood-onset cause of blindness (Delettre, 2002). This implies that normal membrane dynamics are necessary for human mitochondrial function and provides a strong incentive for understanding the underlying biological control of this process (McQuibban, 2006).
The only gene in Drosophila known to be needed specifically for mitochondrial fusion is fuzzy onions (fzo), which regulates mitochondrial fusion only in the male germline of flies (Hales, 1997). In an attempt to extend the genetic analysis of mitochondrial membrane fusion in Drosophila, focus was placed on Rhomboid-7, which is predicted to be mitochondrial by virtue of an N-terminal targeting sequence. When Rhomboid-7 was expressed in mammalian cells, the protein exclusively colocalized with Mitotracker and the F1F0 ATP synthase, respectively, both well-characterized markers for the mitochondrial network. As an initial approach to revealing the function of Rhomboid-7, RNAi was used to disrupt gene expression in cultured Drosophila S2 cells. Treatment with double-stranded (ds) RNA that targets rhomboid-7 resulted in a majority of cells with a highly fragmented mitochondrial network and a quantitative reduction in rhomboid-7 expression. By homology searching, the apparent Drosophila ortholog (CG8479) of mgm1 and OPA1 was identified, that is designate as optic atrophy 1-like (opa1-like) after the mammalian ortholog. Cells treated with dsRNA that targets opa1-like resulted in a fragmented mitochondrial network, a phenotype indistinguishable from rhomboid-7 disruption, and a quantitative reduction in Opa1-like expression. These results suggest that Rhomboid-7 and Opa1-like participate in a similar process of maintaining mitochondrial morphology and that, as in other organisms, disruption of mitochondrial fusion leads to fragmentation. They also suggest that the machinery of membrane fusion control may be conserved between yeast and metazoans (McQuibban, 2006).
To complement the Drosophila RNAi analysis and to investigate whether a similar function was conserved in mammalian cells, mouse mitochondrial rhomboid (called PSARL, presenilins-associated rhomboid-like; Pellegrini, 2001) and mouse OPA1 were expressed in COS-7 cells. Cells that expressed high levels of either PSARL or OPA1 but not a catalytic mutant of PSARL displayed complete aggregation of the mitochondrial network, in a phenotypically identical fashion. This mitochondrial aggregation phenotype resembles the excess fusion caused by overexpression of the mitofusins (Santel, 2001), the mammalian homologs of Fzo. Cells that expressed either PSARL or OPA1 at low levels did not generally disrupt the normal tubular network seen in COS-7 cells. In parallel, the effects were examined of overexpressing Rhomboid-7 in Drosophila S2 cells. As with mammalian cells, this led to mitochondrial aggregation. These data are consistent with the Drosophila RNAi experiments: together, they strongly suggest that, as in yeast, mitochondrial rhomboids and Opa1-like GTPases are involved in the control of mitochondrial membrane dynamics in both flies and mammals (McQuibban, 2006).
Since all other known phenotypes associated with loss of rhomboid-7 or its yeast or mammalian homologs can be attributed to defects in mitochondrial fusion, it is suspected that this is the cause of the abnormal synaptic transmission. There are many studies implicating mitochondria in synaptic function, although their precise role remains to be determined. The current data strongly suggest that fully efficient mitochondrial membrane fusion is necessary for normal photoreceptor activity. Consistent with this, loss of synaptic mitochondria prevents mobilization of the reserve pool of synaptic vesicles. It will be interesting to determine whether impairment of mitochondrial fusion in synapses leads to the same specific defect. In fact, the basic need for mitochondrial fusion and fission in any cells is not fully understood; various theories have been proposed, but the precise reasons for mitochondrial networks being so dynamic remain mysterious (McQuibban, 2006).
Mutations in the human OPA1 gene cause dominant optic atrophy, a common form of childhood-onset blindness. Therefore light-induced photoreceptor degeneration was measured in rhomboid-7 and control flies. Control flies show no defects after 3 days of continuous light. In contrast, over the same period (the maximum length the mutants survive), rhomboid-7pΔ1 mutant photoreceptors degenerated severely, as shown by the almost complete loss of distinct rhabdomeres. This sensitivity to light implies that, in the absence of efficient mitochondrial membrane dynamics, photoreceptor neurons acquire activity-dependent damage at a much higher rate than normal, an interesting possible parallel with human dominant optic atrophy. Importantly, the rhomboid-7pΔ1 mutant eyes showed no detectable degeneration under normal light or dark conditions, and it was these flies that showed the clear defect in signal transfer across the first visual synapse. This suggests that the synaptic defects are primary consequences of Rhomboid-7 loss, and not secondary to photoreceptor loss. Consistent with this conclusion that apoptosis may not be a primary phenotype of Rhomboid-7 loss, Drosophila S2 cells in which rhomboid-7 was reduced by RNAi showed no extra sensitivity to cycloheximide-induced apoptosis (McQuibban, 2006).
The results imply that not all mitochondrial membrane dynamics require functional Rhomboid-7 (otherwise the mutant would be fully lethal, as is the case for the mouse mitofusin genes MFN1 and 2). Instead, Rhomboid-7 is apparently required in places with a high requirement for mitochondrial fusion and/or energy demands. Loss of Opa1-like causes similar phenotypes to loss of Rhomboid-7 which, coupled with what has previously been shown in yeast, strongly suggests that they work in the same processes. It is worth noting, however, that opa1-like mutants are more severe than rhomboid-7 mutants, indicating that Opa1-like has additional functions that are Rhomboid-7 independent. A similar conclusion has been reached in yeast, where loss of Mgm1 causes more severe failure of mitochondrial fusion than loss of Rbd1/Pcp1 (Sesaki, 2003b). One goal of this work was to investigate the consequences of disrupting mitochondrial fusion in metazoans, where the potential for complex phenotypes is greater than in yeast. Although the full mechanisms that underlie the synaptic transmission defects are not know, the results lead to the idea that they are caused by loss of full mitochondrial function at the synapse. The mechanism underlying the striking longevity defect is less obvious but could be related to neural malfunction in regions other than the retina (McQuibban, 2006).
To investigate the requirements for a mitochondrial rhomboid in a multicellular organism, mutations in Drosophila rhomboid-7 were have generated and characterized. The P element {RS3}CB-0229-3 is located in the 5′ UTR of rhomboid-7, thereby potentially disrupting its expression but not the protein coding sequence. Flies homozygous for this insertion are viable and appear healthy but the males are sterile. Precise excision of this P element resulted in reversion to fully wild-type flies, demonstrating that the P element caused the phenotype. This precise excision line is used as a control for subsequent experiments. A predicted null allele of rhomboid-7 was generated by imprecise excision of {RS3}CB-0229-3. The resulting mutation, rhomboid-7pΔ1, lacks the transcriptional start site and the first 18 codons of the protein. In addition to removing the 5′ end of the gene, this deletion disrupts the mitochondrial targeting sequence, so if any residual protein were produced, it would not be targeted appropriately. Although some homozygous adults do emerge, 90% of rhomboid-7pΔ1 flies die before pupariation. Death occurs during both embryonic and larval stages, but those that survive to pupariation develop to adults, although approximately 10% of these die during the process of eclosion, since they get stuck while crawling from the pupal case. Surviving flies appear morphologically normal but all die within 3 days. Males are sterile but the females are fertile. The progeny of homozygous females show exactly the same severity of phenotype as their parental generation and are not rescued by wild-type sperm, demonstrating that there is no maternal or paternal rescue of the homozygous zygotic phenotype. These results indicate that Rhomboid-7 is not essential for Drosophila development but that its absence nevertheless dramatically reduces viability (McQuibban, 2006).
Since surviving males are sterile, the testes of homozygous rhomboid-7pΔ1 mutants were examined and it was found that although there was no gross morphological disruption, the seminal vesicle was small and appeared empty. This was confirmed by dissection: rhomboid-7pΔ1 and P element {RS3}CB-0229-3 mutant testes contained no mature sperm. Significantly, there is an essential mitochondrial fusion process during Drosophila sperm maturation. All the mitochondria in the spermatid coalesce adjacent to the nucleus, then undergo a process of massive membrane fusion. This results in the formation of the nebenkern, a mitochondrial derivative composed of two giant, intertwined mitochondria that eventually unfurl to fill the sperm tail, providing the energy for motility. Nebenkerns are easily seen by phase-contrast microscopy: they appear as phase-dark, round structures of equivalent size and are adjacent to the phase-light nucleus. In contrast to the uniform and regular shapes of control nebenkerns, rhomboid-7pΔ1 mutants and P element {RS3}CB-0229-3 homozygous mutants have irregularly shaped nebenkerns. This phenotype is strikingly similar to that caused by loss of fuzzy onions (fzo1, an ethylmethane sulfonate-induced loss-of-function allele), the only other gene known to be required for mitochondrial membrane fusion in spermatids. This strongly suggests that Rhomboid-7 may also participate in mitochondrial membrane fusion. The structure of rhomboid-7pΔ1 nebenkerns were examined in more detail by transmission electron microscopy. Control nebenkerns show the typical onion-like structure of interleaved coils of membrane. rhomboid-7pΔ1 nebenkerns, however, were composed of many individual mitochondria that had coalesced beside the nucleus, but had failed to fuse, again like fzo1 nebenkerns. These data directly confirm that Rhomboid-7 is required for mitochondrial membrane fusion, at least in the formation of the nebenkern during spermatogenesis (McQuibban, 2006).
By analogy to yeast, it might be expected that Opa1-like would function in regulating mitochondrial membrane dynamics in Drosophila. The phenotype of opa1-like mutants was examined. Two independent P elements inserted into the first and second exons of the opa1-like gene (P{EPgy2}CG8479 and P{lacW}l(2)s3475, respectively) were identified. Both P element lines were early larval lethal. Mitotic clones of opa1-like mutant cells were examined in the male germline to determine whether Opa1-like, like Rhomboid-7 and Fzo, is required for nebenkern membrane fusion. opa1-likeP{lacW}l(2)s3475 and opa1-likeP{EPgy2}CG8479 mutant spermatids have nebenkerns with the same morphological defects as rhomboid-7pΔ1 and fzo1 mutants. These data suggest that Rhomboid-7 and Opa1-like function to regulate the mitochondrial fusion that generates the nebenkern during spermatogenesis (McQuibban, 2006).
Surviving adult rhomboid-7 mutants are morphologically normal, with the exception of a wing-position defect, in which the wings of about 60% of individuals hang down on either side of the abdomen, as opposed to control flies that have their wings tucked and positioned on top of the abdomen. Such defects often indicate flight muscle abnormalities. Examination of the indirect flight muscles of rhomboid-7pΔ1 mutants by light microscopy showed a general disruption of the normal continuous pattern of the phase-dark banding. Examination by electron microscopy showed many small mitochondria in the space between the myofibrils, as compared to larger mitochondria that completely filled the intramyofibril space in control flies. Since muscle maturation in Drosophila involves the fusion of many small mitochondria into larger ones over the first few days of life, these data indicate a role for Rhomboid-7 in mitochondrial fusion in flight muscles as well as spermatids—both tissues with unusual developmental requirements for high levels of fusion (McQuibban, 2006).
rhomboid-7pΔ1 flies live for only 3 days whereas control flies live for an average of 60 days. In addition to this longevity defect, rhomboid-7pΔ1 mutant flies are unable to fly, have extreme difficulty walking, and display erratic twitching in their legs and head. These motor defects could be caused by muscle or neurological disorder (or both), but in most tissues these are difficult to distinguish phenotypically. In order to test specifically whether neuronal activity was affected by rhomboid-7 loss, synaptic transmission was measured in the retina, where muscle defects would be irrelevant, by recording electroretinograms (ERGs; this technique uses electrodes to record depolarisation of neurons in response to light) of rhomboid-7pΔ1 mutants and control flies. By recording extracellular potentials, an average synaptic transmission across the retina is measured. Additionally, intracellular recordings were made to record from single photoreceptors (McQuibban, 2006).
Throughout the stimulation, the responses of control flies showed prominent on- and off-transients; these spikes represent synaptic transmission from R1-6 photoreceptors to large monopolar cells, the first visual interneurons in the optic lobe. The amplitudes of these transients showed no obvious time or intensity dependency. In contrast, both on- and off-transients of rhomboid-7pΔ1 mutants were significantly smaller than those of control flies, with the off-transients showing strong time and intensity dependency. The off-transients to bright and dim pulses died out within 10–20 s and 20–40 s, respectively. Additionally, the graded receptor potential component of the rhomboid-7pΔ1 ERGs was reduced in size during the experiments, much more than that observed with control flies. This was further confirmed by recording intracellular voltage responses of photoreceptors to a brief saturating light pulse. The responses of photoreceptors in rhomboid-7pΔ1 mutants were less than half of those of control photoreceptors, indicating reduced responsiveness to repetitive stimulation. These ERG recordings and intracellular voltage responses indicate that disruption of Rhomboid-7 prevents normal photoreceptor synaptic function, specifically constraining both the generation of light responses and the signal transfer across the first visual synapse (McQuibban, 2006).
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).
Search PubMed for articles about Drosophila rhomboid-7
Cipolat, S., et al. (2006). Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126(1): 163-75. 16839884
Delettre, C., et al. (2002). OPA1 (Kjer type) dominant optic atrophy: a novel mitochondrial disease, Mol. Genet. Metab. 75: 97-107. 11855928
Esser, K., et al. (2002). A novel two-step mechanism for removal of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease Pcp1. J. Mol. Biol. 323(5): 835-43. 12417197
Guan, K., Farh, L., Marshall, T. K. and Deschenes, R. J. (1993). Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene. Curr. Genet. 24: 141-148. 7916673
Hales, K. G. and Fuller, M. T. (1997). Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90: 121-129. 9230308
Herlan, M., et al. (2003). Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA, J. Biol. Chem. 278: 27781-27788. 12707284
Herlan, M., et al. (2004). Alternative topogenesis of Mgm1 and mitochondrial morphology depend on ATP and a functional import motor. J. Cell Biol. 165: 167-173. 15096522
Hermann, G. J., et al. (1998). Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p, J. Cell Biol. 143: 359-373. 9786948
Jeyaraju, D. V., et al. (2006). Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology. Proc. Natl. Acad. Sci. 103(49): 18562-7. 17116872
McQuibban, G. A., Saurya, S. and Freeman, M. (2003). Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423(6939): 537-41. 12774122
McQuibban, G. A., Lee, J. R., Zheng, L., Juusola, M. and Freeman, M. (2006). Normal mitochondrial dynamics requires rhomboid-7 and affects Drosophila lifespan and neuronal function. Curr. Biol. 16(10): 982-9. 16713954
Pellegrini, L. et al. (2001). PAMP and PARL, two novel putative metalloproteases interacting with the COOH-terminus of Presenilin-1 and -2. J. Alzheimers Dis. 3: 181-190. 12214059
Rapaport, D., et al. (1998). Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae. J. Biol. Chem. 273: 20150-20155. 9685359
Santel, A. and Fuller, M. T. (2001). Control of mitochondrial morphology by a human mitofusin. J. Cell Sci. 114: 867-874. 11181170
Sesaki, H., Southard, S. M., Yaffe, M. P. and Jensen, R. E. (2003a). Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane. Mol. Biol. Cell 14(6): 2342-56. 12808034
Sesaki, H., Southard, S. M., Hobbs, A. E. and Jensen, R. E. (2003b). Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner, but remain competent for mitochondrial fusion. Biochem. Biophys. Res. Commun. 308(2): 276-83. 12901865
Sik, A., Passer, B. J., Koonin, E. V. and Pellegrini, L. (2004). Self-regulated cleavage of the mitochondrial intramembrane-cleaving protease PARL yields Pbeta, a nuclear-targeted peptide. J. Biol. Chem. 279(15): 15323-9. 14732705
Tatsuta, T., et al. (2007). m-AAA protease-driven membrane dislocation allows intramembrane cleavage by rhomboid in mitochondria. EMBO J. 26(2): 325-35. 17245427
date revised: 26 January 2007
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