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• W2028567188 abstract "We demonstrate biochemically that the genes identified by sequence similarity as orthologs of the mitochondrial import machinery are functionally conserved in Caenorhabditis elegans. Specifically, tin-9.1 and tin-10 RNA interference (RNAi) treatment of nematodes impairs import of the ADP/ATP carrier into isolated mitochondria. Developmental phenotypes are associated with gene knock-down of the mitochondrial import components. RNAi of tomm-7 and ddp-1 resulted in mitochondria with an interconnected morphology in vivo, presumably due to defects in the assembly of outer membrane fission/fusion components. RNAi of the small Tim proteins TIN-9.1, TIN-9.2, and TIN-10 resulted in a small body size, reduced number of progeny produced, and partial embryonic lethality. An additional phenotype of the tin-9.2(RNAi) animals is defective formation of the somatic gonad. The biochemical demonstration that the protein import activity is reduced, under the same conditions that yield the defects in specific tissues and lethality in a later generation, suggests that the developmental abnormalities observed are a consequence of defects in mitochondrial inner membrane biogenesis. We demonstrate biochemically that the genes identified by sequence similarity as orthologs of the mitochondrial import machinery are functionally conserved in Caenorhabditis elegans. Specifically, tin-9.1 and tin-10 RNA interference (RNAi) treatment of nematodes impairs import of the ADP/ATP carrier into isolated mitochondria. Developmental phenotypes are associated with gene knock-down of the mitochondrial import components. RNAi of tomm-7 and ddp-1 resulted in mitochondria with an interconnected morphology in vivo, presumably due to defects in the assembly of outer membrane fission/fusion components. RNAi of the small Tim proteins TIN-9.1, TIN-9.2, and TIN-10 resulted in a small body size, reduced number of progeny produced, and partial embryonic lethality. An additional phenotype of the tin-9.2(RNAi) animals is defective formation of the somatic gonad. The biochemical demonstration that the protein import activity is reduced, under the same conditions that yield the defects in specific tissues and lethality in a later generation, suggests that the developmental abnormalities observed are a consequence of defects in mitochondrial inner membrane biogenesis. In yeast, the mitochondrial proteome is estimated to contain ∼800 proteins (1Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schonfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13207-13212Crossref PubMed Scopus (703) Google Scholar). In Caenorhabditis elegans, as for most organisms, the mitochondrial genome codes for only 12 of the hundreds of mitochondrial polypeptides, most of which are components of the OXPHOS machinery (2Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar). In addition to ATP production through oxidative phosphorylation, the mitochondrion is a key player in a number of cellular processes including metal ion homeostasis, the synthesis of metabolites, and free radical disproportionation. Ninety eight percent of mitochondrial proteins are translated in the cytosol on free ribosomes and then post- or co-translationally imported into mitochondria. Thus, mitochondrial protein import is a fundamental process in eukaryotic cells (3Baker K.P. Schatz G. Nature. 1991; 349: 205-208Crossref PubMed Scopus (201) Google Scholar, 4Muhlenhoff U. Lill R. Biochim. Biophys. Acta. 2000; 1459: 370-382Crossref PubMed Scopus (178) Google Scholar).The mitochondrion consists of an outer and inner membrane, which serves to separate two aqueous compartments, the matrix and the intermembrane space. There is an elaborate set of proteins within each sub-compartment of the mitochondrion for protein import (5Truscott K.N. Brandner K. Pfanner N. Curr. Biol. 2003; 13: R326-R337Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Curran S.P. Koehler C.M. Koehler C.M. Bauer M.F. Topics in Current Genetics: Mitochondrial Function and Biogenesis. 8. Springer-Verlag, Heidelberg, Germany2004: 59-80Google Scholar). These translocons are specialized and recognize precursors that possess specific targeting and sorting information. In the budding yeast Saccharomyces cerevisiae, the outer mitochondrial membrane contains a single translocase for the passage of polypeptides. The translocase of the outer membrane (TOM) 1The abbreviations used are: TOM, translocase of outer membrane; AAC, ADP/ATP carrier; DHFR, dihydrofolate reductase; TIN/TIM, translocase of inner membrane; DDP, deafness dystonia polypeptide; RNAi, RNA interference; PMSF, phenylmethylsulfonyl fluoride; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; DTCs, distal tip cells; PHB, prohibitin. 1The abbreviations used are: TOM, translocase of outer membrane; AAC, ADP/ATP carrier; DHFR, dihydrofolate reductase; TIN/TIM, translocase of inner membrane; DDP, deafness dystonia polypeptide; RNAi, RNA interference; PMSF, phenylmethylsulfonyl fluoride; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; DTCs, distal tip cells; PHB, prohibitin. complex facilitates translocation across the outer membrane (7Schatz G. J. Biol. Chem. 1996; 271: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 8Paschen S.A. Neupert W. IUBMB Life. 2001; 52: 101-112Crossref PubMed Scopus (75) Google Scholar, 9Pfanner N. Geissler A. Schleiff E. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 339-349Crossref PubMed Scopus (416) Google Scholar). The sorting and assembly machinery, a second complex in the outer membrane, facilitates assembly of outer membrane proteins with complex topologies such as β-barrel structures (10Wiedemann N. Kozjak V. Prinz T. Ryan M.T. Meisinger C. Pfanner N. Truscott K.N. J. Mol. Biol. 2003; 327: 465-474Crossref PubMed Scopus (30) Google Scholar). Once a precursor has passed through the TOM complex to the intermembrane space, the precursor can take one of several routes. Precursors destined for the matrix typically contain an amino-terminal targeting signal that is utilized by the translocase of the inner membrane (TIM)23 complex (11Bauer M.F. Hofmann S. Neupert W. Int. Rev. Neurobiol. 2002; 53: 57-90Crossref PubMed Google Scholar, 12Chacinska A. Pfanner N. Meisinger C. Trends Cell Biol. 2002; 12: 299-303Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The TIM23 translocon facilitates translocation across the mitochondrial inner membrane in a membrane potential dependent manner. Insertion into the inner membrane is accomplished by a related but functionally distinct translocon, the TIM22 complex (5Truscott K.N. Brandner K. Pfanner N. Curr. Biol. 2003; 13: R326-R337Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6Curran S.P. Koehler C.M. Koehler C.M. Bauer M.F. Topics in Current Genetics: Mitochondrial Function and Biogenesis. 8. Springer-Verlag, Heidelberg, Germany2004: 59-80Google Scholar, 13Kaldi K. Neupert W. Biofactors. 1998; 8: 221-224Crossref PubMed Scopus (19) Google Scholar, 14Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Crossref PubMed Scopus (161) Google Scholar). Precursors that utilize the TIM22 translocon such as the carrier family of proteins, Tim22p and Tim23p, have targeting information within the mature polypeptide and lack an amino-terminal targeting sequence. In addition, the TIM22 translocon utilizes two soluble complexes in the intermembrane space, the Tim9p-Tim10p complex and the Tim8p-Tim13p complex, that function to bind to the hydrophobic stretches found in TIM22 substrates as they are escorted across the aqueous intermembrane space (15Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Crossref PubMed Scopus (248) Google Scholar, 16Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 17Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 17: 4816-4822Crossref Scopus (105) Google Scholar, 18Curran S.P. Leuenberger D. Oppliger W. Koehler C.M. EMBO J. 2002; 21: 942-953Crossref PubMed Scopus (158) Google Scholar, 19Curran S.P. Leuenberger D. Schmidt E. Koehler C.M. J. Cell Biol. 2002; 158: 1017-1027Crossref PubMed Scopus (101) Google Scholar).Finally, many aspects of the translocation machinery appear to be conserved in mammalian systems (20Bauer M.F. Gempel K. Reichert A.S. Rappold G.A. Lichtner P. Gerbitz K.D. Neupert W. Brunner M. Hofmann S. J. Mol. Biol. 1999; 289: 69-82Crossref PubMed Scopus (90) Google Scholar, 21Hoogenraad N.J. Ward L.A. Ryan M.T. Biochim. Biophys. Acta. 2002; 1592: 97-105Crossref PubMed Scopus (133) Google Scholar). Mitochondrial dysfunction is a common factor in a broad range of diseases, and numerous mitochondrial proteins have been identified as regulators of the aging process (22Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2587) Google Scholar, 23Lee S.S. Lee R.Y. Fraser A.G. Kamath R.S. Ahringer J. Ruvkun G. Nat. Genet. 2003; 33: 40-48Crossref PubMed Scopus (751) Google Scholar). The first disease Mohr-Tranebjaerg syndrome associated with a defect in protein import is caused by mutations in the intermembrane space import component DDP-1 (deafness dystonia polypeptide) (24Tranebjaerg L. Schwartz C. Eriksen H. Andreasson S. Ponjavic V. Dahl A. Stevenson R.E. May M. Arena F. Barker D. J. Med. Genet. 1995; 32: 257-263Crossref PubMed Scopus (162) Google Scholar, 25Koehler C.M. Leuenberger D. Merchant S. Renold A. Junne T. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2141-2146Crossref PubMed Scopus (268) Google Scholar).To demonstrate the conservation of import mechanisms in higher eukaryotes and to study the developmental consequences of mitochondrial defects, we investigated mitochondrial import in C. elegans. The reverse genetic manipulation used impairs mitochondrial protein translocation in biochemical assays. Gene knock-down of the homologous translocation components led to developmental defects in specific tissues as well. Specifically, knock-down of tin-9.1, tin-9.2, and tin-10 resulted in a small body (Sma) size, reduced brood size, and partial embryonic lethality. tin-9.2(RNAi) animals also had cell migration defects. tomm-7(RNAi) and ddp-1(RNAi) animals displayed defects in mitochondrial morphology. Thus, aspects of human mitochondrial diseases are mimicked in C. elegans and provide an excellent model for understanding the global impact of mitochondrial biogenesis on a multicellular organism.EXPERIMENTAL PROCEDURESStrains and Cloning—Strains were maintained at 20 °C unless otherwise specified by using standard techniques (26Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). Wild-type genes were cloned from N2 Bristol. The mitochondrial outer membrane YFP marker under the expression of the myo-3 promoter was generously donated by the van der Bliek laboratory. Transgenic worms were obtained as described previously (27Labrousse 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 (507) Google Scholar) by injecting expression constructs and the transformation marker, rol-6(su1006), into the gonad of adult wild-type C. elegans.RNAi constructs were created by PCR amplification of genes from either genomic DNA or cDNA pools generated from purified poly(A)+ RNA (Ambion). The PCR products were then cloned into the L4440 vector, and the Escherichia coli host HT115 was used for feeding RNAi studies (28Timmons L. Court D.L. Fire A. Gene (Amst.). 2001; 263: 103-112Crossref PubMed Scopus (1332) Google Scholar). A genomic clone of tin-9.1 and cDNA clones of tomm-7, ddp-1, tin-9.2, and tin-10 were used.Microscopy and Image Analysis—Live worms were mounted on a film of dried agarose in a small volume of M9 medium. The worms were paralyzed with 10 mm aldicarb. Green fluorescent protein variants were viewed with fluorescein isothiocyanate, YFP, or CFP filter sets (Chroma Technologies Corp.). Worms were visualized on a Zeiss Axiovert 200 microscope.RNAi Methods—Eggs were isolated from OP50 fed wild-type worms and allowed to hatch in liquid S-medium overnight in the absence of food. The synchronous L1 larvae were then seeded onto NGM plates containing E. coli expressing double-stranded RNA specific for mitochondrial import components. The plates were incubated at the temperatures stated. At adulthood, worms were moved to fresh RNAi plates and allowed to lay eggs for 4 h. The adult animals were then removed, and the second-generation animals were scored for morphological defects. Third generation animals were obtained in a similar manner.Mitochondria Purification—For large scale RNAi, worms grown in liquid culture were prepared as above except the synchronous and starved L1 larvae were diluted to 2000 worms/ml of S-medium containing freshly induced E. coli expressing dsRNA. The worms were fed until adulthood, and then the animals were cleaned by sucrose flotation and washed thoroughly before utilization for mitochondria purification.Mitochondria were purified from lactate-grown yeast cells (29Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (285) Google Scholar) or from S-medium-cultured adult worms by a modified protocol derived from Jonassen et al. (30Jonassen T. Davis D.E. Larsen P.L Clarke C.F. J. Biol. Chem. 2003; 278: 51735-51742Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The nematodes were resuspended in buffer STEG (250 mm sucrose, 5 mm Tris-HCl, 1 mm EGTA, pH 7.4) with 1 mm PMSF and protease inhibitor mixture (PIC, Roche Diagnostics). The samples were kept cold on ice throughout the fractionation procedure. The animals were homogenized with a Kontes ground glass tissue grinder using 15 strokes. The volume was increased to 25 ml with STEG + 1 mm PMSF/PIC and centrifuged at 750 × g for 10 min. The supernatants were saved, and another 10 ml of STEG +1 mm PMSF, 1× PIC was added to the pellets. The pellets were resuspended and homogenized again with another 15 strokes, and the volumes were increased to 25 ml and centrifuged at 750 × g for 10 min. The supernatants were combined and centrifuged at 12,000 × g for 10 min. The mitochondrial pellets were gently resuspended in 10 ml of STEG without protease inhibitors using a Potter-Elvehjem tissue homogenizer with PTFE pestle (Bellco). The mixture was centrifuged at 750 × g for 10 min. The supernatants were collected, avoiding the pellets, and were centrifuged at 12,000 × g for 10 min. The final mitochondrial pellets were resuspended in STEG. Purified mitochondria were used immediately for in vitro import assays because coupled import-competent organelles could not be recovered after freezing.In Vitro Protein Import Assays—In vitro protein import assays were performed as described (31Rospert S. Schatz G. Celis J.E. 2nd Ed. Cell Biology: A Laboratory Manual. 2. Academic Press, San Diego1998: 277-285Google Scholar). Proteins were synthesized in a rabbit reticulocyte lysate in the presence of [35S]methionine and [35S]cysteine after in vitro transcription of the corresponding gene by SP6 polymerase. The reticulocyte lysate containing the radiolabeled precursor was incubated with isolated mitochondria at the indicated temperatures in import buffer (1 mg/ml bovine serum albumin, 0.6 m sorbitol, 150 mm KCl, 10 mm MgCl2, 2.5 mm EDTA, 2 mm ATP, 2 mm NADH, 20 mm K+ HEPES, pH 7.4). Where indicated, the potential across the mitochondrial inner membrane was dissipated with 1 μm valinomycin. Nonimported radiolabeled proteins were removed by treatment with 100 μg/ml trypsin for 30 min on ice; trypsin was inhibited with 400 μg/ml soybean trypsin inhibitor. Import reactions were separated by SDS-PAGE followed by fluorography. Import was quantified by scanning laser densitometry (Personal Densitometer SI; Amersham Biosciences) and ImageQuant (version 4.2a; Amersham Biosciences).RESULTSWe performed a phylogenetic analysis of five predicted genes in the C. elegans genome with sequence similarity to the small Tim family of proteins and Tom7p with S. cerevisiae, Mus musculus, and Homo sapiens. Specifically, we assembled dendrograms for the annotated genes Y93A3CR.4, C06G3.11, a non-annotated gene found on BAC B0564 (32Bauer M.F. Rothbauer U. Muhlenbein N. Smith R.J. Gerbitz K. Neupert W. Brunner M. Hofmann S. FEBS Lett. 1999; 464: 41-47Crossref PubMed Scopus (65) Google Scholar), Y66D12A.22, and ZK652.2 here referred to as ddp-1, tin-9.1, tin-9.2, tin-10, and tomm-7, respectively (Fig. 1A); trees were assembled using ClustalW (33Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55203) Google Scholar). Similar to other eukaryotic systems, the C. elegans genome codes for at least one member of each small Tim family: one from the Tim8 family, two Tim9 isoforms, one Tim10/12 family member, and one from Tim13. Therefore, C. elegans shares a similar complement of components in the conserved TIM22 pathway for the import of polytopic inner membrane proteins.The tin-9.2 gene had not been annotated previously, although an SL1 spliced tin-9.2 sequence is present on cDNA EST yk1059h04.5. To confirm that the gene product of this cDNA could be translated and was correctly targeted to the mitochondria, we placed the cDNA clone upstream of and inframe with CFP under the control of the myo-3 gene promoter for expression in muscle cells. This construct was co-injected with a plasmid bearing the rol-6(su1006) mutation which causes the animals carrying the transgenes to move twisting in a circle rather than crawling. TIN-9.2::CFP accumulated in tubular structures in the muscle cells of transgenic animals (Fig. 1B) and co-localized with the mitochondria-specific dye rhodamine 6G (data not shown). Thus, TIN-9.2 localizes to mitochondria as expected.Isolation of Import-competent Mitochondria from C. elegans—Gene knock-down of the translocation components was obtained by RNAi treatment. The transcript levels for the specific genes were down-regulated in RNAi-treated but not control animals when assessed by quantification of RT-PCR products (data not shown). To establish the effectiveness of RNAi treatment and to demonstrate that the C. elegans homologs function as they do in yeast, we developed a protocol that yields import-competent mitochondria. The mitochondria were purified from adult C. elegans essentially as described previously (34Jonassen T. Marbois B.N. Faull K.F. Clarke C.F. Larsen P.L. J. Biol. Chem. 2002; 277: 45020-45027Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). However, C. elegans are grown in a medium containing high levels of Ca2+ that can disrupt mitochondrial function, so we substituted the Ca2+-chelator EGTA for EDTA in the isolation buffer. In organello import assays are well established for fungi and have been instrumental for the fundamental understanding of mitochondrial assembly in a single cell (31Rospert S. Schatz G. Celis J.E. 2nd Ed. Cell Biology: A Laboratory Manual. 2. Academic Press, San Diego1998: 277-285Google Scholar). [35S]Methionine and [35S]cysteine precursors for porin, ADP/ATP carrier (AAC), and the synthetic precursor pSu9-DHFR were synthesized in vitro for in organello import assays (see “Experimental Procedures”). The established yeast assay uses porin to test for import into the mitochondrial outer membrane. A membrane potential is not required for this translocation. For all import assays nonimported precursor is removed by protease treatment. Porin was imported into the mitochondrial outer membrane in both nematode and control yeast mitochondria (Fig. 2). Insertion into the outer membrane was confirmed by alkali extraction and flotation through a sucrose density gradient (35Allen R. Egan B. Gabriel K. Beilharz T. Lithgow T. FEBS Lett. 2002; 514: 347-350Crossref PubMed Scopus (42) Google Scholar).Fig. 2Mitochondria isolated from wild-type adults are import-competent. [35S]Methionine- and [35S]cysteine-radiolabeled precursors, porin, AAC, and pSu9-DHFR, were synthesized in a reticulocyte lysate system and incubated with coupled mitochondria isolated from C. elegans or S. cerevisiae. Equal aliquots of the import reaction were removed at the specified time points and treated with trypsin to remove nonimported precursor. The membrane potential (ΔΨ) was dissipated with valinomycin treatment. Porin insertion into the outer membrane was confirmed by carbonate extraction and sucrose flotation (35Allen R. Egan B. Gabriel K. Beilharz T. Lithgow T. FEBS Lett. 2002; 514: 347-350Crossref PubMed Scopus (42) Google Scholar). As a control, an aliquot of the porin import reaction was treated with 0.1% Triton X-100. Inner membrane insertion of AAC was confirmed by extraction with 0.1 m Na2CO3, pH 11.0 (36Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1380) Google Scholar). The Su9-DHFR precursor (p) was processed to the mature (m) form when imported into the mitochondrial matrix in the presence of ΔΨ. Reactions were separated by SDS-PAGE and visualized by fluorography. As a standard (S), 10% of the translation reaction added to mitochondria was included.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The established yeast assay uses the AAC to test for import into the mitochondrial inner membrane. Succinate was added to the mitochondria to maintain a membrane potential and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone and K+ ionophore valinomycin were added to dissipate the membrane potential. The inner membrane substrate AAC was imported into the inner membrane of the purified C. elegans mitochondria in the presence of a membrane potential (ΔΨ) (Fig. 2). The insertion was confirmed by alkali extraction (36Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1380) Google Scholar). The synthetic precursor pSu9-DHFR was imported into the matrix of the purified C. elegans mitochondria in a ΔΨ-dependent manner, as it is in yeast, and the presequence was cleaved by the matrix-processing protease (37Isaya G. Kalousek F. Fenton W.A. Rosenberg L.E. J. Cell Biol. 1991; 113: 65-76Crossref PubMed Scopus (103) Google Scholar). In sum, these in organello import assays in samples from C. elegans render this system amenable to the study of mitochondrial protein import.Mitochondria from Worms with RNAi-mediated Gene Knockdown of tin-9.1 and tin-10 Are Defective in Inner Membrane Protein Import—In yeast, Tim9p and Tim10p are essential proteins involved in the TIM22 import pathway that facilitates the insertion of polytopic inner membrane proteins, such as AAC (14Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Crossref PubMed Scopus (161) Google Scholar, 18Curran S.P. Leuenberger D. Oppliger W. Koehler C.M. EMBO J. 2002; 21: 942-953Crossref PubMed Scopus (158) Google Scholar). To test whether the nematode homologs TIN-9.1 and TIN-10 also mediate the import of AAC, we used feeding RNAi to down-regulate TIN-9.1 and TIN-10 in C. elegans, and subsequently mitochondria were purified from treated adults. As expected, pSu9-DHFR import and maturation were similar in the RNAi-treated and wild-type mitochondria (Fig. 3, top panel). However, the import of AAC was decreased in mitochondria isolated from tin-9.1(RNAi) and tin-10(RNAi) worms by 76 and 81%, respectively, in comparison to mitochondria from control adults (Fig. 3, bottom panel). This severe decrease suggests that TIN-9.1 and TIN-10 are functionally conserved components of the TIM22 import pathway. In addition, these biochemical studies demonstrate that the RNAi treatment is successful for decreasing TIN-9.1 and TIN-10 expression levels.Fig. 3RNAi of TIN-9.1 and TIN-10 results in an import defect specific for the inner membrane substrate AAC. Import reactions were performed as in Fig. 2, except that mitochondria were isolated from N2 worms fed E. coli expressing dsRNA for tin-9.1 or TIN-10 or no dsRNA (control). Import was quantitated by scanning laser densitometry, and the % import was normalized to the longest time point for control mitochondria in the presence of ΔΨ. STD, a standard of 10% of the translation reaction added to mitochondria; p, precursor; m, mature.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phenotypes Associated with Gene Knock-down of Mitochondrial Import Components—To better understand the role that mitochondrial protein translocation has on the development of a multicellular organism, we utilized RNAi to disrupt the function of TIN-9.1 and TIN-10 as in Fig. 3, as well as TOMM-7, DDP-1, and TIN-9.2. Decreases in nuclear encoded mitochondria-associated gene function lead to an increase in life span (23Lee S.S. Lee R.Y. Fraser A.G. Kamath R.S. Ahringer J. Ruvkun G. Nat. Genet. 2003; 33: 40-48Crossref PubMed Scopus (751) Google Scholar, 38Dillin A. Hsu A.L. Arantes-Oliveira N. Lehrer-Graiwer J. Hsin H. Fraser A.G. Kamath R.S. Ahringer J. Kenyon C. Science. 2002; 298: 2398-2401Crossref PubMed Scopus (774) Google Scholar, 39Hekimi S. Guarente L. Science. 2003; 299: 1351-1354Crossref PubMed Scopus (376) Google Scholar) and decreased fertility (30Jonassen T. Davis D.E. Larsen P.L Clarke C.F. J. Biol. Chem. 2003; 278: 51735-51742Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 40Tsang W.Y. Lemire B.D. Biochim. Biophys. Acta. 2003; 1638: 91-105Crossref PubMed Scopus (91) Google Scholar). The mean life span of tomm-7(RNAi), ddp-1(RNAi), and tin-9.1(RNAi) animals showed no significant difference compared with control animals (Fig. 4). The mean life span of tin-9.2(RNAi) and tin-10(RNAi) animals was decreased 10 and 18%, respectively. We interpret this decrease in life span to be a result of sickness, rather than accelerated aging because these animals exhibit slower movement and increased incidence of protruding vulva (Pvl) phenotypes.Fig. 4RNAi of mitochondrial import components does not influence life span. Eggs from N2 OP50 fed worms were isolated and allowed to hatch on plates seeded with E. coli expressing dsRNA specific for mitochondria translocation components. The plates were incubated at 25 °C and monitored every 24 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess fertility, we determined the brood size and the fraction of embryonic lethality over three sequential generations (Table I). tomm-7(RNAi), ddp-1(RNAi), and control animals showed similar brood sizes and no embryonic lethality in all generations tested. In contrast, tin-9.1(RNAi) worms showed a decrease in average brood size of 51% in the first generation compared with control animals, 38% in the second generation, and 39% in the third generation. tin-10(RNAi) worms also had smaller brood sizes, and this phenotype was more penetrant than in tin-9.1(RNAi) worms in subsequent generations. Unlike the other translocation components tested, tin-10(RNAi) worms had decreased brood sizes and increased embryonic lethality in the second generation, and the third generation animals that grew to adulthood were sterile. First generation tin-9.2(RNAi) worms showed no defects in brood size. However, the subsequent F2 and F3 progeny had average brood sizes decreased by 59 and 47%, respectively, compared with control animals. The RNAi animals that presented a sterile phenotype were not included in the calculation of the average brood size. The seemingly high standard deviations recorded for the observed phenotypes probably result in part from the combined occurrence of decreased fertility and embryonic lethality. Most interestingly, there were shared reproductive defects in the second and third generation associated with tin-9.1(RNAi), tin-9.2(RNAi), and tin-10(RNAi) worms, which are presumed to function together in the import pathway for polytopic inner membrane proteins. The decrease in viable eggs and larvae is expected, because the homologous genes are essential for viability in yeast.Table IDevelopmental effects of RNAi for mitochondrial import componentsTreatmentaE. coli strain HT115(DE3) expressing either no dsRNA from the empty vector L4440 (control) or dsRNA from vector L4440 encoding specific mitochondrial translocation components, tomm-7, ddp-1, tin-9.1, tin-9.2, or tin-10, were fed to strain N2F1F2F3Average brood sizebBrood sizes were scored for 20 individual F2 and F3 worms given a tin-9.1, tin-9.2, or tin-10 RNAi E. coli food source. Of these 20, (n) developed into fertile adult hermaphrodites. Sets less than 20 are due to arrest of the animal at a sexually immature stage or complete sterility% embryonic lethalAverage brood sizebBrood sizes were scored for 20 individual F2 and F3 worms given a tin-9.1, tin-9.2, or tin-10 RNAi E. coli food source. Of these 20, (n) developed into fertile adult hermaphrodites. Sets less than 20 are due to arrest of the animal at a sexually immature stage or complete sterility% embryonic lethalAverage brood sizebBrood sizes were scored for 20" @default.
• W2028567188 created "2016-06-24" @default.
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• W2028567188 creator A5009462050 @default.
• W2028567188 creator A5030553063 @default.
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• W2028567188 date "2004-12-01" @default.
• W2028567188 modified "2023-10-18" @default.
• W2028567188 title "Defective Mitochondrial Protein Translocation Precludes Normal Caenorhabditis elegans Development" @default.
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