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J. Biol. Chem., Vol. 278, Issue 34, 32091-32099, August 22, 2003
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The Mitochondrial Prohibitin Complex Is Essential for Embryonic Viability and Germline Function in Caenorhabditis elegans*
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From the
Swammerdam Institute for Life Sciences,
Section for Molecular Biology, University of Amsterdam, Kruislaan 318,
Amsterdam 1098 SM, The Netherlands, ¶Canadian
Institutes of Health Research, Membrane Protein Research Group, Department of
Biochemistry, University of Alberta, Edmonton T6G 2H7, Canada, and
Nijmegen Center for Mitochondrial
Disorders, University Medical Center Nijmegen, Geert Grooteplein 10, Nijmegen
6500 HB, The Netherlands
Received for publication, May 9, 2003 , and in revised form, June 5, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prohibitins (Phb1p and Phb2p), referred to here as PHB proteins, are evolutionarily strongly conserved proteins that are located in mitochondria in yeast, plants, and mammals (1–6). In mammalian and yeast cells, it has been demonstrated that PHB proteins associate with each other to form a high molecular weight complex (the PHB complex) in the mitochondrial inner membrane (5, 6). Although diverse cellular functions have been attributed to both PHB proteins (see Ref. 7 for a review), such as a role in cell cycle regulation (8–11) and in cell surface signaling (12, 13), these functions are difficult to reconcile with the exclusive localization of mammalian PHB proteins to mitochondria (1, 3). To date, studies on the yeast PHB complex have provided convincing evidence for a direct role in mitochondrial function. The PHB complex has been found to co-purify with the m-AAA (matrix-ATPase associated with a variety of cellular activities) protease, and a role as a negative regulator of the protease has been proposed (5). Yeast PHB proteins are capable of stabilizing newly synthesized mitochondrially encoded proteins through direct interaction, suggesting a role in mitochondrial respiratory complex assembly (5, 6). We have suggested a role for PHB proteins in the biogenesis of mitochondria as a holdase/unfoldase type of protein specifically required in situations of metabolic stress (6). Based on structural data from chemical cross-linking and mass spectrometry, we predict a barrel-like structure for the yeast PHB complex, in the cavity of which mitochondrial products might be held (14).
At the phenotypic level, disruption of PHB genes in yeast results in a shortening of the replicative life span due to premature aging (3). This shortening of life span contrasts with the lack of an observable growth phenotype under laboratory conditions. However, deletion of PHB genes is lethal in combination with mutations of the mitochondrial inheritance machinery (4), of the AAA-mitochondrial proteases (5), or of the mitochondrial phosphatidylethanolamine biosynthetic machinery (15). The lack of a clear growth phenotype in yeast PHB mutants might reflect a redundancy in assembly factors. Alternatively, PHB mutations may have a stronger phenotype in organisms or tissues with a greater dependence on mitochondrial energy generation. In support of this latter hypothesis, deletion of a PHB homologue in Drosophila melanogaster results in lethality during larval development (16), suggesting that PHB proteins are essential during one or more steps in the differentiation of multicellular organisms.
In this study, we have used Caenorhabditis elegans as a model organism to study the role of PHB proteins during organismal development. First, we demonstrate by blue native electrophoresis (BNE)1 that PHB proteins in the nematode form a high molecular weight complex in the mitochondrial membrane similar to that observed in yeast and humans (6). Second, by using RNA-mediated interference (RNAi), we monitor the effects of depleting PHB proteins at different developmental stages. Depletion of PHB proteins during embryogenesis results in developmental arrest. When PHB levels are depleted during postembryonic development, several somatic and germline effects are observed. Germline defects range from sterility to severely reduced brood sizes with a high incidence of embryonic lethality of the progeny. Somatic defects include a reduced body size and a morphologically abnormal somatic gonad. A direct link to mitochondrial dysfunction is demonstrated by the severely altered mitochondrial morphology observed in body wall muscle cells of phb(RNAi) animals. We find a slightly but significantly reduced oxygen consumption rate in phb(RNAi)-treated worms compared with control worms. In addition, we show that PHB protein contents are elevated in situations of altered mitochondrial metabolism, such as when imbalances in respiratory enzyme subunits occur, as has been reported in other systems (6, 17, 18). In strong contrast to the yeast situation, we report a severe loss of function phenotype for depletion of the mitochondrial PHB complex during organismal development. Our results show that C. elegans serves as a useful model organism for the study of mitochondrial metabolism and mitochondrial biogenesis and in better understanding mitochondrial diseases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Strains and Conditions—Worms were cultured at room temperature (
22 °C) unless otherwise noted
(19). C. elegans
strains used were N2 Bristol wild-type strain and
glo-1(zu391). Sequence Comparisons and Alignments—Percentage identities and similarities between human, C. elegans, and S. cerevisiae PHB proteins were obtained from the Incyte Genomics database. Human PHB1 protein shares 66/83 and 50/68% identity/similarity with the predicted C. elegans genes Y37E3.9 and T24H7.1, respectively. Y37E3.9 shares 53/79 and 50/71% identity/similarity with the yeast PHB1 and PHB2 proteins, respectively, whereas T24H7.1 shares 53/75% and 48/71% identity/similarity with the yeast PHB2 and PHB1 proteins, respectively.
Amino acid sequences were obtained from SWISS-PROT and TrEMBL. Accession numbers were as follows: PHB_HUMAN, Swiss-Prot P35232 [GenBank] ; PHB2_HUMAN, TrEMBL Q99623 [GenBank] ; Y37E3.9, TrEMBL Q9BKU4; T24H7.1, Swiss-Prot P50093 [GenBank] . GenBankTM accession numbers were as follows: PHB_HUMAN, NP_002625 [GenBank] .1; PHB2_HUMAN, NP_009204 [GenBank] ; Y37E3.9, AAK27865 [GenBank] .1; T24H7.1, AAA68353 [GenBank] .1. Multiple sequence alignments were performed using ClustalW.
RNA Interference—For phb-2(RNAi) a 1.9-kb PCR product, amplified using N2 genomic DNA as a template with primers Phb2-F (5'-CGCATGGTATTTCCTGAGTAGG-3') and Phb2-R (5'-CTCTCTTCAAAATGCCAACCC-3'), was digested with BstBI and SalI, and the 1.06-kb fragment was cloned into XhoI/ClaI-digested pBluescript II SK and pBC-KS (PDI BioScience, Aurora, Canada) to place the gene fragment under the control of the T7 promoter in both the forward and reverse orientations. The constructs were co-transformed into the Escherichia coli strain BL21-SI (Invitrogen), which contains an integrated T7 RNA polymerase gene under control of the salt-inducible proU promoter. Bacteria were grown overnight at 37 °C in LBON medium (1% bactotryptone, 0.5% yeast extract) containing appropriate antibiotics. Expression of constructs was induced with 0.2 M NaCl. For phb-1(RNAi), a 0.8-kb fragment was amplified using N2 genomic DNA as template with primers Phb1-F (5'-CAATGTTGATGGAGGTCAACG-3') and Phb1-R (5'-GGTGACATTCTTGTTCTTGGC-3'). A 0.627-kb SacI/EcoRV fragment was cloned into likewise digested pBluescript II SK and pBC-KS. Empty vectors were used as controls.
N2 L4 hermaphrodites were fed bacteria expressing sense and antisense RNAs or bacteria containing empty vectors on plates containing 0.2 M salt. Animals were transferred onto freshly seeded plates every 24 h. Offspring were analyzed 24 h after transfer. For monitoring postembryonic development, gravid adults were bleached, and the embryos were allowed to hatch overnight on unseeded plates. Starved L1 larvae were transferred to plates containing 0.2 M salt and seeded with bacteria expressing PHB-RNAs or containing empty vectors.
RT-PCR—Total RNA from embryos was prepared as follows.
Approximately 60 gravid hermaphrodites were picked when RNAi showed the
highest effectiveness (after 72 h of feeding) from salt-induced vector control
plates or from salt-induced RNAi plates and dissolved in a 1:10 solution of
bleach in 1 M NaOH. Embryos were collected and washed twice in 1 ml
of phosphate-buffered saline, pelleted, and resuspended in 200 µl of lysis
buffer (0.5% SDS, 5% 
-mercaptoethanol, 10 mM EDTA, 10
mM Tris-HCl, pH 7.5, 0.5 mg/ml proteinase K). Samples were
incubated at 55 °C for 1 h and processed further using TRIzol Reagent
(Invitrogen) following the manufacturer's instructions. RNA pellets were
dissolved in 100 µl of diethylpyrocarbonate-treated H2O. For
each RT-PCR, 2 µl of RNA was used.
RT-PCR was performed using the SuperScriptTM One-StepTM RT-PCR system (Invitrogen), following the manufacturer's instructions. To test the efficiency of the RNAi treatment by RT-PCR, reactions were prepared from control and phb-2(RNAi)-treated embryos. phb-1 (Y37E3.9) and phb-2 (T24H7.1) RNA levels were normalized using the isocitrate dehydrogenase gene (F35G12.2). After 30 cycles of amplification, products were analyzed by agarose gel electrophoresis. Primers used for RT-PCR analysis were designed using the predicted cDNA sequences: Idh-F, 5'-AGCAACGTCCTCGGTCATAC-3'; Idh-R, 5'-GATGAACGCAGTTGGATTGG-3'; Phb1-F, 5'-CAATGTTGATGGAGGTCAACG-3'; Phb1-R, 5'-GGTGACATTCTTGTTCTTGGC-3'; Phb2-F, 5'-GGACACCGAGCTATCATGTTC-3'; Phb2-R, 5'-CAACATCAATCCTCCTGTTGG-3'. For phb-1(RNAi), RT-PCR was performed on RNA isolated from the same number of young adults fed dsRNA from the L1 stage.
Electrophoresis and Western Blot—For BNE/two-dimensional PAGE, mitochondrial membrane fractions were prepared as described (20) with minor modifications. Briefly, young adult worms were collected by centrifugation at 1,500 x g for 10 min and washed with M9 buffer three times and once with MSM-E buffer (21). The pellet was resuspended in 10% (w/v) MSM-E buffer containing a proteinase-inhibitor mixture (Roche Applied Science), and cells were disrupted with a Teflon homogenizer. The lysate was centrifuged at 1,500 x g for 10 min at 4 °C. The supernatant was centrifuged at 23,500 x g for 30 min, and the pellet containing submitochondrial particles was resuspended in BNE extraction buffer containing 1.5% lauryl maltoside. Two-dimensional PAGE was performed as described (22) using 14% SDS-polyacrylamide gels.
For one-dimensional SDS-PAGE, worm pellets were resuspended in 5 volumes of SDS-sample buffer, boiled for 5 min, and the proteins were resolved on 10% gels (23). Following electrophoresis, proteins were blotted to nitrocellulose, and immunoreactive material was visualized by chemiluminescent detection (ECLTM; Amersham Biosciences) according to the manufacturer's instructions.
Pharyngeal Pumping and Defecation Measurements—Synchronized animals were placed on nematode growth media plates containing 0.2 M salt seeded with BL21-SI bacteria producing phb-1 or phb-2 RNAs or containing empty vectors and raised at 15, 20, or 25 °C. Animals were scored for pharyngeal pumping and defecation in their first day of fertility. To score fertility, 70 L4-staged animals were transferred individually to fresh plates. Statistical analysis was performed using analysis of variance tests.
Oxygen Consumption Rate—Oxygen consumption rates were measured as previously described (24) using a Clark-type electrode (Rank Bros. Ltd., Bottisham, Cambridge, UK) with some minor modifications. Young adult worms fed either RNA-expressing bacteria or control bacteria were washed and collected in S-basal buffer. Approximately 100 µl of slurry pellet of worms were delivered into the chamber in 3 ml of S-basal medium. The chamber was kept at 25 °C, and measurements were done for 5–15 min, depending on the oxygen consumption rate. The slope of the straight portion of the plot was used to derive the oxygen consumption rate. Worms were recovered after respiration measurements and collected for protein quantification. Rates were normalized to protein content. We performed 10 independent measurements per strain. Statistic analysis was performed with an analysis of variance test followed by a least square deviation post hoc test.
Mitochondrial Morphology—Mitochondrial morphology was monitored in glo-1(zu391) mutant worms, which have little or no gut granule autofluorescence2 expressing the Pmyo-3::mito::GFP construct (25) and the transformation marker pRF4 rol-6(su1006). Morphology was monitored by laser-scanning confocal microscopy.
Doxycycline Treatment—Synchronized L1 hermaphrodites were transferred to plates containing 40 µg/ml doxycycline. Worms were collected after 72 h for Western blot analysis.
Antibodies—A polyclonal antibody raised against the 25
C-terminal amino acids of the murine PHB1 protein has been previously
described (3). Polyclonal
antibody against the yeast subunit of F1-ATPase was a gift from Prof.
Jan Berden. Anti-actin antibody was obtained from ICN (clone C4) and used at a
dilution of 1:10.000.
Microscopy—Animals were mounted on 2% agarose pads and observed under a Zeiss Axioskop-2 research microscope with a SPOT-2 digital camera (Carl Zeiss Canada Ltd., Calgary, Canada) or a Zeiss Axiocam camera. GFP images were acquired with a Zeiss LSM 510 confocal microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C. elegans Contains a Conserved PHB Complex in the Mitochondrial Inner Membrane—The C. elegans sequence data base contains two predicted genes, Y37E3.9 and T24H7.1, having extensive sequence identity with the yeast PHB1 and PHB2 genes (26). Amino acid sequence comparisons with the human and yeast PHB proteins identify Y37E3.9 and T24H7.1 as being orthologs of PHB1 and PHB2, respectively (see "Materials and Methods" for details). A sequence alignment of human and C. elegans PHB proteins is shown in Fig. 1A. We propose to name Y37E3.9 phb-1 and T24H7.1 phb-2 in order to follow the previously proposed convention (7). For the corresponding proteins, we will use the nomenclature PHB-1 and PHB-2. When necessary, gene and protein names can be preceded by letters specifying the species (e.g. cephb-1 and cePHB-1 for the C. elegans gene and protein, respectively).
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It has been previously shown that together the PHB proteins form a high molecular weight complex with an estimated size of 1 MDa in the mitochondrial inner membrane of yeast and humans (6). To determine the size of the C. elegans PHB complex, mitochondrial membrane extracts were resolved by two-dimensional gel electrophoresis (22). In the first dimension, membrane protein complexes are separated by blue native electrophoresis according to their size. In the second dimension, denaturing SDS-PAGE separates protein complexes into their subunits (Fig. 2A). Western blots of this gel were immunostained with the polyclonal antibody raised against the C terminus (last 25 amino acids) of the murine PHB1 protein (APP-2) (3). As seen in Fig. 2B, the PHB1 antibody cross-reacts with bands of 30 and 32 kDa after separation in the second dimension, the lower immunoreactive band (30 kDa) being of higher intensity. The predicted sizes for cePHB-1 (275 amino acids) and cePHB-2 (286 amino acids) are 30 and 31.8 kDa, respectively. Because the preimmune serum does not recognize these bands (data not shown) and the migration of the protein complex in the first dimension is similar to the migration of the yeast and human PHB complexes (6), we believe the 30- and 32-kDa immunoreactive bands correspond to cePHB-1 and cePHB-2 (additional evidence is presented below; see "Discussion"). This demonstrates that in C. elegans the PHB proteins form a large mitochondrial complex similar to PHB complexes of other systems (6).
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The TMHMM algorithm (27) suggests a transmembrane helix for cePHB-2 (positions 12–34), whereas no transmembrane region is predicted for cePHB-1. This is in agreement with topology predictions for the yeast PHB proteins (14). The yeast PHB proteins are imported into mitochondria without the cleavage of an N-terminal leader peptide (6). Given that the observed protein sizes on SDS-PAGE correlate well with the predicted molecular weight of the mature proteins and given the sequence homology with yeast (data not shown), it is tempting to believe that the C. elegans PHB proteins are also imported to mitochondria without cleavage of a large N-terminal leader peptide and that the predicted transmembrane region of cePHB-2 is present in the mature protein as incorporated in the complex.
PHB Proteins Are Essential during Embryonic Development—RNAi is the phenomenon in which introduction of dsRNA results in potent and specific inactivation of the corresponding gene through the degradation of endogenous mRNA (28, 29). RNAi mediated by the ingestion of bacteria producing sense and antisense RNAs has proven to be a powerful tool in the analysis of gene function in C. elegans (30, 31).
We performed RNAi by feeding worms E. coli engineered to express
sense and antisense RNAs corresponding to the predicted exon-rich genomic
sequences of both cephb-1 and cephb-2
(Fig. 1B). L4-staged
hermaphrodites were placed onto plates seeded with bacteria producing
phb-dsRNA or bacteria containing empty vectors. The hermaphrodites
were transferred to fresh seeded plates every 24 h, and the progeny remaining
on the plate were scored 24 h later. RNAi for phb-1, phb-2, or
phb-1+2 resulted in 60–70% embryonic arrest
(Fig. 3A). Brood sizes
of phb(RNAi) animals were slightly reduced compared with controls.
Embryonic lethality was scored under salt-induced and noninduced conditions.
No embryonic arrest was observed with vector controls
(Fig. 3A) or in the
absence of salt (data not shown).
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The development of affected embryos was followed by light microscopy. A developmental delay was observed in RNAi-treated embryos when compared with controls (Fig. 3B). Developmental arrest ranged from the gastrula to the 1.5-fold stages (data not shown). This variability may reflect differences in the efficiency of RNAi in each embryo.
PHB Proteins Are Required for Germline Function—RNAi allows the postembryonic depletion of gene transcripts essential for embryonic development. When L1-staged larvae were fed phb-1 and/or phb-2 dsRNA, they showed a delay in development. Control worms reached adulthood 48 h after starting feeding and laid most of their offspring between 48 and 72 h, whereas phb(RNAi)-treated worms initiated egg laying after 72 h and only reached their peak of fertility between 72 and 96 h. Between 30 and 40% of phb-2(RNAi)-treated animals develop into sterile adults. Fertile adults had severely reduced brood sizes with a high incidence of mortality in the progeny (Fig. 4A). The postembryonic phenotypes were comparable for phb-1(RNAi), phb-2(RNAi), or phb-1+phb-2(RNAi) animals.
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RNAi-treated hermaphrodites were examined under the microscope, and somatic defects including a reduced body size and abnormal gonad morphology were observed. Most prominent are the reduced number of germ nuclei and their abnormal morphologies (Fig. 4B, c and f) and defective oogenesis (Fig. 4B, b, c, d, f, h, and i) and spermatogenesis that probably are responsible for the animals' sterility or severely reduced fertility. Accumulation of unfertilized oocytes in the uterus can also be seen (Fig. 4B, e). Abnormal embryos were also observed (data not shown).
Specificity and Effectiveness of the RNAi Treatment—We analyzed the specificity and effectiveness of the RNAi treatment by RT-PCR and immunostaining. Embryos were collected from gravid hermaphrodites after feeding on phb-2 dsRNA for 48 h and RNA was extracted. The RT-PCR results show that phb-2 mRNA is greatly depleted in the RNAi-treated embryos, whereas mRNAs for phb-1 or for F35G12.2, a control gene encoding the mitochondrial NAD+-isocitrate dehydrogenase were present at levels comparable with untreated embryos (Fig. 5A). To test the specificity and efficiency of the phb-1-RNAi treatment during larval growth, L1-staged animals were fed phb-1 dsRNA and allowed to develop into young adults. The adults were collected and their RNA extracted. The RT-PCR results show that phb-1 mRNA is severely depleted, whereas mRNAs for phb-2 and for F35G12.2 are present at wild type levels (Fig. 5B). To analyze the effects of RNAi on PHB protein levels, mitochondria were isolated from young adults fed either RNAi-expressing bacteria or control bacteria from the L1 stage. Mitochondrial membrane proteins from control RNAi worms, from phb-1(RNAi), and from phb-2(RNAi) worms were resolved by BNE/two-dimensional gel electrophoresis. Western blot analysis shows a clear reduction in the amounts of the two PHB proteins in both RNAi treatments (Fig. 5C), whereas ATP synthase levels were essentially unchanged. These results are in agreement with observations in yeast, where the disruption of either phb gene leads to the loss of both proteins, although the mRNA for the intact gene can still be detected (4). As in yeast (4, 6), the C. elegans PHB-1 and PHB-2 subunits are interdependent for assembly and protein stability. These results indicate that RNAi for both phb-1 and phb-2 genes is specific and effective.
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Although the cephb-1 and cephb-2 genes share 64% nucleotide sequence identity, their mRNAs are not subject to RNAi cross-interference, further emphasizing the specificity and effectiveness of the RNAi treatment.
Physiological Rhythms and Temperature Sensitivity of phb(RNAi) Mutants—We investigated whether pharyngeal pumping rates or the defecation cycle is affected in phb(RNAi)-treated worms. Pharyngeal pumping of RNAi-treated animals was more irregular and significantly reduced (p < 0.05) at 15, 20, and 25 °C when compared with the control (Table I and data not shown). Similarly, the defecation cycles of phb(RNAi) animals at 20 °C were slightly but significantly (p < 0.05) prolonged when compared with wild type worms (Table I).
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The observed postembryonic phenotypes, developmental delay, reduced body size, decreased fertility, and slowed pharyngeal pumping and defecation, are consistent with phb(RNAi) animals being metabolically compromised. To further address this possibility, we assessed the effects of growth at higher temperatures, which may aggravate any metabolic defects due to increased metabolic rates (32). The fraction of sterile animals significantly increases with temperature, from 10% at 15 °C to 46% at 20 °C to 70% at 25 °C for phb-2(RNAi) and from 6% at 15 °C to 8% at 20 °C to 32% at 25 °C for phb-1(RNAi). Moreover, at 25 °C, fertile phb(RNAi) hermaphrodites showed a significantly reduced brood size when compared with RNAi animals grown at 20 °C (data not shown).
Altered Mitochondrial Respiration and Morphology in phb(RNAi) Mutants—To analyze the effect of depletion of PHB proteins on mitochondrial function, we measured oxygen consumption rates of phb(RNAi) animals (see "Materials and Methods" for details). As shown in Fig. 6, the oxygen consumption rates are slightly but significantly reduced when compared with control worms (p = 0.048 and p = 0.027 for phb-1(RNAi) and phb-2(RNAi) worms, respectively), but RNAi-treated worms did not differ in their respiration rate (p = 0.787).
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Mitochondrial morphology changes are often associated with compromised electron transport and ATP synthesis (33). The C. elegans strain glo-1(zu391) lacks autofluorescence and birefringent gut granules, making it ideal for the analysis of GFP expression.2 We used a glo-1(zu391) strain expressing a mitochondrially targeted GFP fusion protein expressed from the muscle-specific myo-3 promoter (25). Body wall muscle mitochondria in control worms appear tubular, elongated, and well structured, running parallel to the body axis and often parallel to the myofibrils (Fig. 7A) (25). In contrast, in phb(RNAi)-treated animals, muscle mitochondria appear fragmented and disorganized (Fig. 7, B and C).
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phb(RNAi) Animals Show Increased Sensitivity to Oxidative Stress—The PHB complex has been suggested to play a role in the assembly of oxidative phosphorylation complexes (6). Deficiencies in the mitochondrial respiratory chain can lead to the increased production of reactive oxygen species (ROS) and increased sensitivity to oxidative stress. We grew control and phb-2(RNAi)-treated worms in the presence of increasing concentrations of paraquat, which triggers the production of superoxide radicals. phb-2(RNAi) animals are markedly more sensitive to paraquat than control worms (Fig. 8). This suggests that a deficiency in PHB proteins may result in an increase in the rate of free radical production, making the animals less able to deal with an externally applied oxidative stress, in a decreased ability to detoxify free radicals, or in both.
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Induction of PHB Proteins upon Mitochondrial Stress—PHB protein expression increases when there is an imbalance in the synthesis of mitochondrial respiratory chain enzyme subunits in yeast (6, 17) and in human cells (18).
To test this in C. elegans, synchronized L1 larvae were treated with 40 µg/ml doxycycline, a specific inhibitor of mitochondrial translation (34), and allowed to develop to the late L3/L4 stage, where they arrest, and their PHB protein levels were analyzed by immunodetection. Doxycycline slows worm development and, at concentrations equal to or above 60 µg/ml, causes developmental arrest at the L3 stage (35). In four independent experiments, the increase in PHB protein levels varied between 30 and 40% after 72 h of treatment when compared with controls (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously reported that the yeast PHB complex stabilizes newly synthesized mitochondrial translation products by transiently interacting with them and have suggested a role in the assembly of mitochondrial respiratory chain (MRC) complexes (6). Prohibitin disruption in yeast results in a reduction in its replicative life span, plausibly due to a slight but cumulative decline in cellular metabolic capacity (3). In contrast, disruption of the Drosophila homologue of prohibitin is lethal during passage from larva to pupa (16), suggesting a strong dependence of these differentiating cells on prohibitin function.
To further investigate this, we set out to study the effect of lack of prohibitins during the development of Caenorhabditis elegans. C. elegans contains two predicted genes that we have named phb-1 and phb-2. RT-PCR analysis of phb-1+2(RNAi)-treated worms shows that both genes are expressed (Fig. 5, A and B). We also demonstrate by blue native electrophoresis that the gene products form a complex in the mitochondrial inner membrane as in other organisms studied. Moreover, the fact that the PHB complex is depleted in either phb-1(RNAi) or phb-2(RNAi) animals, as seen by BNE analysis (Fig. 5C), demonstrates that in C. elegans PHB-1 and PHB-2 are interdependent at the level of protein complex formation, as we and others have previously reported in yeast (4, 6) and humans (18).
The PHB-1 polyclonal antiserum (APP-2) used in this study was raised
against the carboxyl-terminal 25 amino acids of the murine protein
(3). The APP-2 antibody
recognizes two bands of 30 and 32 kDa in C. elegans,
corresponding to the predicted protein sizes of the mature proteins. The
antibody recognizes with higher affinity the 30-kDa band (PHB-1). We believe
that those bands correspond to the cePHB-1 and cePHB-2 proteins for the
following reasons: 1) the immunoreactive bands are present in mitochondrial
membrane extracts and co-migrate as a high molecular weight complex in the
first dimension of BNE analysis (Fig.
2B) as observed in other systems; 2) the APP-2 preimmune
sera recognize neither of the two bands (data not shown); and 3) both bands
are up-regulated after doxycycline treatment (data not shown), a treatment
that has also been shown to increase levels of PHB proteins in mammals
(18) and is similar to
situations described in yeast
(6). And finally and most
convincingly, specific down-regulation of the immunoreactive bands occurs
after specifically down-regulating phb-1 or phb-2 gene
expression (Fig.
5C).
The phb-2(RNAi) effect was slightly stronger than that of the phb-1(RNAi) (see Figs. 3A and 4A, Table I, and the percentages of sterile animals observed at different temperatures). This is probably due to the fact that RNAi efficiency can vary depending on the length of the dsRNA; notice that the genomic DNA fragment cloned for phb-2 covers a larger exonic region. Also to be noticed is the fact that postembryonic RNAi is usually less effective than embryonic RNAi, since the interference effect is diluted out as the worm grows in size. Consistent with this, a higher amount of phb-1 mRNA is detected in RT-PCR analysis of phb-1(RNAi) mutants after postembryonic treatment (Fig. 5A) when compared with the amount of phb-2 mRNA detected in RT-PCR analysis of phb-2(RNAi) mutant embryos (Fig. 5B). Both of these phenomena might contribute to the different effectiveness observed between A and B of Fig. 5.
The RNA interference experiments indicate that the mitochondrial PHB
complex is essential for embryonic development and required for germline
differentiation in the nematode. The most severe phenotype we observed in
phb(RNAi)-treated worms during postembryonic development is
sterility. Since RNAi can often mimic a null phenotype, it is tempting to
speculate that a total lack of PHB proteins during postembryonic development
will also lead to sterility. Functional genomic analysis by systematic RNAi
shows that genes involved in basal metabolic processes account for 50% of
genes producing embryonic arrest or sterility phenotypes
(30). Therefore, our
observations indicate that PHB proteins serve an essential role in cellular
energy metabolism during organismal development.
The phenotypes observed in C. elegans strongly indicate that prohibitins are specifically required during cellular proliferation. Embryonic and germline cells are relatively undifferentiated and highly proliferative. Because the germline is the only proliferating tissue during adulthood, it may require higher metabolic activity and thus be more susceptible to the loss of function of PHB proteins than other somatic tissues. This is in agreement with previous observations in mammalian cells. It is known that prohibitins are more expressed in proliferating than in nonproliferating cells of the same type, and mammalian tumor cells have increased levels of PHB proteins (18). In testis, PHB proteins are expressed during times of proliferation/differentiation but are lost entirely from the mature sperm (18, 36, 37). Therefore, proliferating undifferentiated cells might be particularly susceptible to the lack of prohibitins. Consistent with it, up-regulation of PHB proteins occurs during cell cycle entry (18).
We find a strong effect on mitochondrial morphology when prohibitin expression is reduced. Mitochondria change shape during cell division, during differentiation, and in response to diverse cellular cues. Body wall muscle cells, although differentiated, increase considerably in volume during larval development; thus, the number of mitochondria also increases per cell. This finding shows that PHB proteins play an important role in mitochondrial biogenesis. Recent reports have shown that worms with defective electron transport or ATP synthesis also have altered mitochondrial morphologies (33), linking mitochondrial function to the regulation of mitochondrial biogenesis. The observations that yeast prohibitin mutants are synthetically lethal with mitochondrial morphology mutants (4) and with the phosphatidylethanolamine biosynthetic machinery (15) strongly suggest that the prohibitin complex has an important role in establishing or maintaining the integrity of mitochondrial membranes. Here, we demonstrate that the lack of prohibitins affects mitochondrial distribution and morphology in body wall muscle cells. This situation might become particularly deleterious during cellular proliferation/differentiation, since cells need to achieve the required amount and distribution of mitochondria in order to fulfill their energy requirements. Consistent with this, PHB proteins are essential during embryogenesis and germline differentiation. The altered mitochondrial morphology/distribution, together with the observed reduction in oxygen consumption rate of phb(RNAi) mutants, provides a direct link between PHB deficiency and mitochondrial dysfunction. Although the differences in oxygen consumption rates are small, they are reproducible and statistically significant. Moreover, RNAi does not result in a total removal of the proteins. Therefore, differences can be expected to be larger in the total absence of the PHB complex. In addition, it may well be that the effect is more apparent in tissues that rely more on mitochondrial energy generation, and measuring oxygen consumption in the whole worm might mask a stronger effect on those tissues.
The absence of a holdase/unfoldase protein that ensures correct assembly of MRC enzymes will result in improperly folded mitochondrial subunits that cannot be correctly assembled into functional respiratory enzymes. Hydrophobic proteins may accumulate in the membrane and cause proton leakage and damage to the membrane. ROS production may also increase, leading to MRC dysfunction. All of this together will ultimately compromise cellular metabolic efficiency. The results we obtained are consistent with phb(RNAi) animals being metabolically compromised; they develop more slowly and have reduced body size, fecundity, and physiological rhythms. Moreover, we detect a reduced oxygen consumption rate in phb(RNAi) animals and a strong defect in mitochondrial morphology. At this stage, we do not know whether simply a decline in cellular metabolic capacity can account for the altered mitocondrial morphology observed in phb(RNAi) mutants or, alternatively, PHB proteins play a more direct role in mitochondrial membrane stability, and further investigation in this field is required. Furthermore, in support of the hypothesis that lack of PHB complex might indirectly increase ROS damage (by increased ROS production and/or increased ROS sensitivity), we found that phb(RNAi) animals are markedly more sensitive to exogenously added free radicals than wild type worms.
PHB proteins are specifically required in situations of mitochondrial stress. PHB mRNA levels are induced in yeast cells at the diauxic shift, when cells switch from nonoxidative to oxidative growth (38). Similarly, expression levels of PHB proteins increase in yeast mutants where imbalances between nuclear and mitochondrially encoded subunits occur (6, 17). Inhibition of mitochondrial protein synthesis also leads to imbalances between mitochondrial and nuclear-encoded gene products (39). Indeed, the presence of mitochondrial translation inhibitors results in an increase in PHB protein levels in cultured human cells (18) and in C. elegans (this work). The temperature sensitivity of the phenotype could also indicate a higher requirement for PHB proteins, since metabolic demands increase with temperature (32). Alternatively, temperature might directly or indirectly affect the stability or turnover of mitochondrial respiratory complexes or the PHB complex.
Data from genome-wide analysis of gene expression in C. elegans (40) indicate that phb-2 (T24H7.1) is expressed at all developmental stages, with increased expression in the proliferating embryo as compared with the four-cell stage embryo. Another peak of expression is observed at the L2/L3 larval stage, when gonad proliferation starts. This expression pattern fits with the phenotypes observed in phb(RNAi) animals. Additionally, in aged adult hermaphrodites, no significant expression of phb-2 is detected, being in agreement with the decreased levels of prohibitins observed during senescence (18).
Mitochondria and a functional MRC are essential to fulfill the energy requirements during nematode growth and development (35). An energy-related developmental checkpoint at the L3 to L4 transition has been proposed in situations of seriously impaired MRC function (41). The C. elegans RNAi phenotypes for prohibitins resemble those of known MRC genes (35) (see also RNAi phenotypes for mev-1 and isp-1 on the World Wide Web at www.wormbase.org/). Therefore, it will be interesting to determine whether phb null mutants can be maternally rescued and develop to the L3 stage as seen with the null MRC mutants (35) and to determine how hypomorphic phb mutations will affect nematode growth and development. Fully understanding the scope and severity of effects on mitochondrial function arising from the lack of prohibitins will help to further elucidate the molecular mechanism of action of the PHB complex and its precise role in mitochondrial biogenesis.
For the first time, our investigations have addressed the effects of the loss of the PHB complex at different developmental stages of a complex multicellular organism. We demonstrate by RNAi that the mitochondrial PHB complex is essential for embryonic development, strongly suggesting that a PHB null mutation will be lethal. We also demonstrate that PHB proteins are necessary for normal mitochondrial morphology and respiration. We predict that mutations in either of the human phb genes may be responsible for some mitochondrial diseases that have yet to be described at a molecular level.
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FOOTNOTES |
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* This work was supported by European Commission Grant QLG1-CT-2001-00966. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| Present address: EMBO, Meyerhofstrasse I, D-69117 Heidelberg, Germany.
** These two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 31-20-5257921; Fax:
31-20-5257924; E-mail:
artal{at}science.uva.nl.
1 The abbreviations used are: BNE, blue native electrophoresis; RNAi,
RNA-mediated interference; RT, reverse transcription; dsRNA, double-stranded
RNA; GFP, green fluorescent protein; ROS, reactive oxygen species; MRC,
mitochondrial respiratory chain.
2 G. J. Hermann and J. R. Priess, unpublished observations.
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ACKNOWLEDGMENTS |
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We thank Philip J. Coates for enlightening discussions and Alexander van der Bliek, Greg J. Hermann, and James R. Priess for the glo-1(zu391) strain expressing Pmyo-3::mito::GFP. Celine Moorman, Sander van der Linden, Nadine Vastenhouw, and members of Ronald H. A. Plasterk's laboratory are thanked for kind help. We thank Piet van Egmond for helping with the oxygraph; Marian de Jong for technical support; Wijnand Takkenberg, Erik Manders, and Julio Mateos Langerak for assistance in confocal microscopy; and Sara Magalhaes and Marta Montserrat for assistance in statistical analysis.
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