Effects of Mutation or Deletion

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-7^pΔ1, 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-7^pΔ1 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-7^pΔ1 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-7^pΔ1 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-7^pΔ1 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-7^pΔ1 nebenkerns were examined in more detail by transmission electron microscopy. Control nebenkerns show the typical onion-like structure of interleaved coils of membrane. rhomboid-7^pΔ1 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-like^{P{lacW}l(2)s3475} and opa1-like^{P{EPgy2}CG8479} mutant spermatids have nebenkerns with the same morphological defects as rhomboid-7^pΔ1 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-7^pΔ1 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-7^pΔ1 flies live for only 3 days whereas control flies live for an average of 60 days. In addition to this longevity defect, rhomboid-7^pΔ1 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-7^pΔ1 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-7^pΔ1 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-7^pΔ1 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-7^pΔ1 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).

Reference names in red indicate recommended papers.

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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