The Mitochondrial Prohibitin Complex Is Essential for Embryonic Viability and Germline Function in Caenorhabditis elegans
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m304877200
ISSN1083-351X
AutoresMarta Artal‐Sanz, William Y.W. Tsang, Esther Willems, Les Grivell, Bernard D. Lemire, Hans van der Spek, Leo Nijtmans,
Tópico(s)ATP Synthase and ATPases Research
ResumoProhibitins 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. 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. 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 (1Ikonen E. Fiedler K. Parton R.G. Simons K. FEBS Lett. 1995; 358: 273-277Crossref PubMed Scopus (168) Google Scholar, 2Snedden W.A. Plant Mol. Biol. 1997; 33: 753-756Crossref PubMed Scopus (59) Google Scholar, 3Coates P.J. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar, 4Berger K.H. Yaffe M.P. Mol. Cell. Biol. 1998; 18: 4043-4052Crossref PubMed Scopus (160) Google Scholar, 5Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar, 6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). 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 (5Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar, 6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). Although diverse cellular functions have been attributed to both PHB proteins (see Ref. 7Nijtmans L.G. Artal S.M. Grivell L.A. Coates P.J. Cell Mol. Life Sci. 2002; 59: 143-155Crossref PubMed Scopus (250) Google Scholar for a review), such as a role in cell cycle regulation (8McClung J.K. Danner D.B. Stewart D.A. Smith J.R. Schneider E.L. Lumpkin C.K. Dell'Orco R.T. Nuell M.J. Biochem. Biophys. Res. Commun. 1989; 164: 1316-1322Crossref PubMed Scopus (144) Google Scholar, 9Nuell M.J. Stewart D.A. Walker L. Friedman V. Wood C.M. Owens G.A. Smith J.R. Schneider E.L. Dell' Orco R. Lumpkin C.K. Danner D.B. McClung J.K. Mol. Cell. Biol. 1991; 11: 1372-1381Crossref PubMed Scopus (228) Google Scholar, 10Wang S. Nath N. Adlam M. Chellappan S. Oncogene. 1999; 18: 3501-3510Crossref PubMed Scopus (204) Google Scholar, 11Wang S. Nath N. Fusaro G. Chellappan S. Mol. Cell. Biol. 1999; 19: 7447-7460Crossref PubMed Scopus (142) Google Scholar) and in cell surface signaling (12Terashima M. Kim K.-M. Takahiro Adachi e. Lamers M.C. EMBO J. 1994; 13: 3782-3792Crossref PubMed Scopus (206) Google Scholar, 13Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (242) Google Scholar), these functions are difficult to reconcile with the exclusive localization of mammalian PHB proteins to mitochondria (1Ikonen E. Fiedler K. Parton R.G. Simons K. FEBS Lett. 1995; 358: 273-277Crossref PubMed Scopus (168) Google Scholar, 3Coates P.J. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (5Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar). Yeast PHB proteins are capable of stabilizing newly synthesized mitochondrially encoded proteins through direct interaction, suggesting a role in mitochondrial respiratory complex assembly (5Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar, 6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). 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 (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). 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 (14Back J.W. Sanz M.A. De Jong L. De Koning L.J. Nijtmans L.G. De Koster C.G. Grivell L.A. Van Der Spek H. Muijsers A.O. Protein Sci. 2002; 11: 2471-2478Crossref PubMed Scopus (142) Google Scholar). At the phenotypic level, disruption of PHB genes in yeast results in a shortening of the replicative life span due to premature aging (3Coates P.J. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (4Berger K.H. Yaffe M.P. Mol. Cell. Biol. 1998; 18: 4043-4052Crossref PubMed Scopus (160) Google Scholar), of the AAA-mitochondrial proteases (5Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar), or of the mitochondrial phosphatidylethanolamine biosynthetic machinery (15Birner R. Nebauer R. Schneiter R. Daum G. Mol. Biol. Cell. 2003; 14: 370-383Crossref PubMed Scopus (79) Google Scholar). 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 (16Eveleth Jr., D.D. Marsh J.L. Nucleic Acids Res. 1986; 14: 6169-6183Crossref PubMed Scopus (70) Google Scholar), 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) 1The 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.1The 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. that PHB proteins in the nematode form a high molecular weight complex in the mitochondrial membrane similar to that observed in yeast and humans (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). 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 (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar, 17Nijtmans L.G. Artal Sanz M. Bucko M. Farhoud M.H. Feenstra M. Hakkaart G.A. Zeviani M. Grivell L.A. FEBS Lett. 2001; 498: 46-51Crossref PubMed Scopus (59) Google Scholar, 18Coates P.J. Nenutil R. McGregor A. Picksley S.M. Crouch D.H. Hall P.A. Wright E.G. Exp. Cell Res. 2001; 265: 262-273Crossref PubMed Scopus (167) Google Scholar). 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. Strains and Conditions—Worms were cultured at room temperature (∼22 °C) unless otherwise noted (19Lewis J.A. Fleming J.T. Methods Cell Biol. 1995; 48: 3-29Crossref PubMed Scopus (493) Google Scholar). 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; PHB2_HUMAN, TrEMBL Q99623; Y37E3.9, TrEMBL Q9BKU4; T24H7.1, Swiss-Prot P50093. GenBank™ accession numbers were as follows: PHB_HUMAN, NP_002625.1; PHB2_HUMAN, NP_009204; Y37E3.9, AAK27865.1; T24H7.1, AAA68353.1. 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% β-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 SuperScript™ One-Step™ 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 (20Ishii N. Fujii M. Hartman P.S. Tsuda M. Yasuda K. Senoo-Matsuda N. Yanase S. Ayusawa D. Suzuki K. Nature. 1998; 394: 694-697Crossref PubMed Scopus (575) Google Scholar) with minor modifications. Briefly, young adult worms were collected by centrifugation at 1,500 × g for 10 min and washed with M9 buffer three times and once with MSM-E buffer (21Kayser E.B. Morgan P.G. Hoppel C.L. Sedensky M.M. J. Biol. Chem. 2001; 276: 20551-20558Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). 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 × g for 10 min at 4 °C. The supernatant was centrifuged at 23,500 × 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 (22Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1882) Google Scholar) 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 (23Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). Following electrophoresis, proteins were blotted to nitrocellulose, and immunoreactive material was visualized by chemiluminescent detection (ECL™; 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 (24Braeckman B.P. Houthoofd K. De Vreese A. Vanfleteren J.R. Mech. Ageing Dev. 2002; 123: 105-119Crossref PubMed Scopus (100) Google Scholar) 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 autofluorescence 2G. J. Hermann and J. R. Priess, unpublished observations. expressing the Pmyo-3::mito::GFP construct (25Labrousse A.M. Zappaterra M.D. Rube D.A. van der Bliek A.M. Mol. Cell. 1999; 4: 815-826Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar) 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 (3Coates P.J. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar). Polyclonal antibody against the yeast β 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. 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 (26The C. elegans Sequencing ConsortiumScience. 1998; 282: 2012-2018Crossref PubMed Scopus (3548) Google Scholar). 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 (7Nijtmans L.G. Artal S.M. Grivell L.A. Coates P.J. Cell Mol. Life Sci. 2002; 59: 143-155Crossref PubMed Scopus (250) Google Scholar). 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). 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 (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). To determine the size of the C. elegans PHB complex, mitochondrial membrane extracts were resolved by two-dimensional gel electrophoresis (22Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1882) Google Scholar). 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) (3Coates P.J. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar), 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 (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). The TMHMM algorithm (27Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (8747) Google Scholar) 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 (14Back J.W. Sanz M.A. De Jong L. De Koning L.J. Nijtmans L.G. De Koster C.G. Grivell L.A. Van Der Spek H. Muijsers A.O. Protein Sci. 2002; 11: 2471-2478Crossref PubMed Scopus (142) Google Scholar). The yeast PHB proteins are imported into mitochondria without the cleavage of an N-terminal leader peptide (6Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (444) Google Scholar). 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 (28Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11430) Google Scholar, 29Bass B.L. Cell. 2000; 101: 235-238Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). 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 (30Fraser A.G. Kamath R.S. Zipperlen P. Martinez-Campos M. Sohrmann M. Ahringer J. Nature. 2000; 408: 325-330Crossref PubMed Scopus (1356) Google Scholar, 31Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., and Ahringer, J. (2001) Genome Biol., 2, Research: 0002.1–0002.10Google Scholar). 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). 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. 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 (F
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