Mitochondrial Fatty Acid Synthesis Type II: More than Just Fatty Acids
2008; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.r800068200
ISSN1083-351X
AutoresJ. Kalervo Hiltunen, Melissa S. Schonauer, Kaija J. Autio, Telsa M. Mittelmeier, Alexander J. Kastaniotis, Carol L. Dieckmann,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoEukaryotes harbor a highly conserved mitochondrial pathway for fatty acid synthesis (FAS), which is completely independent of the eukaryotic cytosolic FAS apparatus. The activities of the mitochondrial FAS system are catalyzed by soluble enzymes, and the pathway thus resembles its prokaryotic counterparts. Except for octanoic acid, which is the direct precursor for lipoic acid synthesis, other end products and functions of the mitochondrial FAS pathway are still largely enigmatic. In addition to low cellular levels of lipoic acid, disruption of genes encoding mitochondrial FAS enzymes in yeast results in a respiratory-deficient phenotype and small rudimentary mitochondria. Recently, two distinct links between mitochondrial FAS and RNA processing have been discovered in vertebrates and yeast, respectively. In vertebrates, the mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase and the RPP14 subunit of RNase P are encoded by the same bicistronic transcript in an evolutionarily conserved arrangement that is unusual for eukaryotes. In yeast, defects in mitochondrial FAS result in inefficient RNase P cleavage in the organelle. The intersection of mitochondrial FAS and RNA metabolism in both systems provides a novel mechanism for the coordination of intermediary metabolism in eukaryotic cells. Eukaryotes harbor a highly conserved mitochondrial pathway for fatty acid synthesis (FAS), which is completely independent of the eukaryotic cytosolic FAS apparatus. The activities of the mitochondrial FAS system are catalyzed by soluble enzymes, and the pathway thus resembles its prokaryotic counterparts. Except for octanoic acid, which is the direct precursor for lipoic acid synthesis, other end products and functions of the mitochondrial FAS pathway are still largely enigmatic. In addition to low cellular levels of lipoic acid, disruption of genes encoding mitochondrial FAS enzymes in yeast results in a respiratory-deficient phenotype and small rudimentary mitochondria. Recently, two distinct links between mitochondrial FAS and RNA processing have been discovered in vertebrates and yeast, respectively. In vertebrates, the mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase and the RPP14 subunit of RNase P are encoded by the same bicistronic transcript in an evolutionarily conserved arrangement that is unusual for eukaryotes. In yeast, defects in mitochondrial FAS result in inefficient RNase P cleavage in the organelle. The intersection of mitochondrial FAS and RNA metabolism in both systems provides a novel mechanism for the coordination of intermediary metabolism in eukaryotic cells. Mitochondrial research has been enjoying a renaissance during the last two decades because of major discoveries of previously unknown or overlooked processes such as mitochondrial fusion and fission, mechanisms and regulation of transcription and translation, iron-sulfur cluster biogenesis, structure and assembly of respiratory chain complexes, mitochondria-to-nucleus "retrograde" signaling, mechanisms of mitochondrial inheritance, programmed cell death, and the role of mitochondria in human disease. This review focuses on the process of fatty acid biosynthesis discovered relatively recently in mitochondria.The de novo synthesis of fatty acids in eukaryotes can take place in at least two subcellular compartments: in the cytoplasm (FAS 2The abbreviations used are: FAS, fatty acid synthesis; ACP, acyl carrier protein; PDH, pyruvate dehydrogenase. type I) and in mitochondria (FAS type II). Type II synthesis has its genesis in bacteria and is also found in plant chloroplasts (1White S.W. Zheng J. Zhang Y.M. Rock C.O. Annu. Rev. Biochem. 2005; 74: 791-831Crossref PubMed Scopus (601) Google Scholar). Why has the bacterial type FAS pathway been maintained in mitochondria, when the "classic" cytoplasmic pathway provides most of the cellular fatty acids? Surprisingly, respiratory competence in yeast is dependent on the ability of mitochondria to synthesize fatty acids.Mitochondria in almost all eukaryotic organisms contain their own genome encoding a small number of primarily hydrophobic subunits of the respiratory chain complexes and ATP synthase (2Burger G. Gray M.W. Lang B.F. Trends Genet. 2003; 19: 709-716Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). The genes are transcribed mainly in one or two long transcripts in many organisms from mammals to Schizosaccharomyces pombe (reviewed in Refs. 3Clayton D.A. Annu. Rev. Biochem. 1984; 53: 573-594Crossref PubMed Scopus (326) Google Scholar and 4Schafer B. Gene (Amst.). 2005; 354: 80-85Crossref PubMed Scopus (47) Google Scholar) or as several multigenic transcripts such as in Saccharomyces cerevisiae (reviewed in Ref. 5Dieckmann C.L. Staples R.R. Int. Rev. Cytol. 1994; 152: 145-181Crossref PubMed Scopus (77) Google Scholar). tRNA processing is essential for the expression of mitochondrial mRNAs because tRNAs are interspersed between the mRNAs in the genome-long vertebrate precursor RNA (3Clayton D.A. Annu. Rev. Biochem. 1984; 53: 573-594Crossref PubMed Scopus (326) Google Scholar) and are also co-transcribed with many of the Saccharomyces protein-encoding mRNAs (5Dieckmann C.L. Staples R.R. Int. Rev. Cytol. 1994; 152: 145-181Crossref PubMed Scopus (77) Google Scholar). RNase P is responsible for the endonucleolytic cleavages at the 5′-ends of the mature tRNAs (6Hollingsworth M.J. Martin N.C. Nucleic Acids Res. 1987; 15: 8845-8860Crossref PubMed Scopus (13) Google Scholar), whereas a distinct tRNA endonuclease frees the 3′-ends (7Chen J.Y. Martin N.C. J. Biol. Chem. 1988; 263: 13677-13682Abstract Full Text PDF PubMed Google Scholar). We have shown that fatty acid biosynthesis in mitochondria is linked to RNase P expression in vertebrates (8Autio K.J. Kastaniotis A.J. Pospiech H. Miinalainen I.J. Schonauer M.S. Dieckmann C.L. Hiltunen J.K. FASEB J. 2008; 22: 569-578Crossref PubMed Scopus (44) Google Scholar) and assembly and/or activity in Saccharomyces (9Schonauer M.S. Kastaniotis A.J. Hiltunen J.K. Dieckmann C.L. Mol. Cell. Biol. 2008; 28: 6646-6657Crossref PubMed Scopus (39) Google Scholar).An intersection of two pathways frequently provides a point for controlling metabolic fluxes. In some systems, intersecting pathways are switched on or off in parallel, whereas in others, the pathways are regulated reciprocally. Our hypothesis is that the intersection of the mitochondrial FAS II pathway with RNA processing has been maintained throughout evolution as a means to regulate mitochondrial function relative to the nutritional state of the cell. The details of the intersection in yeast and vertebrates are distinct. The mitochondrial FAS pathway in yeast controls the maturation or activity of mitochondrial RNase P, which cleaves the 5′-leaders of mitochondrial precursor tRNAs (9Schonauer M.S. Kastaniotis A.J. Hiltunen J.K. Dieckmann C.L. Mol. Cell. Biol. 2008; 28: 6646-6657Crossref PubMed Scopus (39) Google Scholar). In humans and in other vertebrates as well, a nuclear bicistronic mRNA encodes both the RPP14 subunit of RNase P and 3-hydroxyacyl-ACP dehydratase (HTD2) in the FAS II pathway (8Autio K.J. Kastaniotis A.J. Pospiech H. Miinalainen I.J. Schonauer M.S. Dieckmann C.L. Hiltunen J.K. FASEB J. 2008; 22: 569-578Crossref PubMed Scopus (44) Google Scholar).Mitochondrial FAS Enzymes and PathwaysThe cytosolic FAS type I pathway comprises one (a homodimer of α2-subunits in higher eukaryotes) or two (a heterododecamer of α6β6-subunits in fungi) multifunctional polypeptides, whereas the mitochondrial pathway comprises independent monofunctional polypeptides that carry out individual steps in FAS (Fig. 1). The first component identified in the mitochondrial pathway was isolated from Neurospora crassa. Labeling with [14C]pantothenic acid led to the identification of an ACP (10Brody S. Mikolajczyk S. Eur. J. Biochem. 1988; 173: 353-359Crossref PubMed Scopus (53) Google Scholar), and it was shown subsequently that the Neurospora protein is indeed involved in de novo synthesis of fatty acids in mitochondria (11Mikolajczyk S. Brody S. Eur. J. Biochem. 1990; 187: 431-437Crossref PubMed Scopus (61) Google Scholar). Other eukaryotes were also shown to contain a mitochondrial form of ACP (12Chuman L. Brody S. Eur. J. Biochem. 1989; 184: 643-649Crossref PubMed Scopus (50) Google Scholar). The initial identification of mitochondrial ACP as a structural component of the membrane-bound bovine mitochondrial respiratory Complex I was puzzling (13Runswick M.J. Fearnley I.M. Skehel J.M. Walker J.E. FEBS Lett. 1991; 286: 121-124Crossref PubMed Scopus (157) Google Scholar), but it was later shown that mammalian mitochondria also contain a soluble form of ACP (14Cronan J.E. Fearnley I.M. Walker J.E. FEBS Lett. 2005; 579: 4892-4896Crossref PubMed Scopus (60) Google Scholar).Since the initial discovery of mitochondrial ACP, the mitochondrial FAS pathway has been well characterized, particularly in S. cerevisiae. This model organism does not contain Complex I but expresses mitochondrial ACP (encoded by the ACP1 gene) and the entire remaining retinue of enzymes required for the synthesis of saturated fatty acids in mitochondria. The yeast malonyl-CoA:ACP transferase (Mct1) (15Schneider R. Brors B. Burger F. Camrath S. Weiss H. Curr. Genet. 1997; 32: 384-388Crossref PubMed Scopus (66) Google Scholar), phosphopantetheine transferase (Ppt2) (16Stuible H.P. Meier S. Wagner C. Hannappel E. Schweizer E. J. Biol. Chem. 1998; 273: 22334-22339Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), ketoacyl synthase (Cem1) (17Harington A. Herbert C.J. Tung B. Getz G.S. Slonimski P.P. Mol. Microbiol. 1993; 9: 545-555Crossref PubMed Scopus (57) Google Scholar), and ketoacyl reductase (Oar1) (15Schneider R. Brors B. Burger F. Camrath S. Weiss H. Curr. Genet. 1997; 32: 384-388Crossref PubMed Scopus (66) Google Scholar) are very similar to their prokaryotic counterparts, whereas the yeast HFA1 gene has been found to encode a mitochondrial acetyl-CoA carboxylase homologous to cytosolic Acc1 (18Hoja U. Marthol S. Hofmann J. Stegner S. Schulz R. Meier S. Greiner E. Schweizer E. J. Biol. Chem. 2004; 279: 21779-21786Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The yeast proteins that catalyze the final two steps of the fatty acid elongation cycle, Htd2 and 2-enoyl-ACP reductase (Etr1), do not have clear sequence similarities to known prokaryotic FAS type II enzymes. Mutations in the yeast HTD2 gene were identified through a plasmid-loss, colony-sectoring screen in which the complementing Escherichia coli homolog was expressed on a plasmid (19Kastaniotis A. Autio K. Sormunen R. Hiltunen J. Mol. Microbiol. 2004; 53: 1407-1421Crossref PubMed Scopus (67) Google Scholar). Our search for 2-enoyl-CoA reductase activity in yeast led to the identification of mitochondrial Etr1 in Candida tropicalis and its homolog in S. cerevisiae, Etr1/Mrf1′ (20Torkko J.M. Koivuranta K.T. Miinalainen I.J. Yagi A.I. Schmitz W. Kastaniotis A.J. Airenne T.T. Gurvitz A. Hiltunen K.J. Mol. Cell. Biol. 2001; 21: 6243-6253Crossref PubMed Scopus (63) Google Scholar). In contrast to an initial observation of nuclear localization for Etr1 (21Yamazoe M. Shirahige K. Rashid M.B. Kaneko Y. Nakayama T. Ogasawara N. Yoshikawa H. J. Biol. Chem. 1994; 269: 15244-15252Abstract Full Text PDF PubMed Google Scholar), a series of subsequent experiments demonstrated that the bulk of the protein was localized to the mitochondrial matrix (20Torkko J.M. Koivuranta K.T. Miinalainen I.J. Yagi A.I. Schmitz W. Kastaniotis A.J. Airenne T.T. Gurvitz A. Hiltunen K.J. Mol. Cell. Biol. 2001; 21: 6243-6253Crossref PubMed Scopus (63) Google Scholar). Etr1 displayed enoyl thioester reductase activity and was necessary to support the growth of yeast cells on a non-fermentable carbon source (20Torkko J.M. Koivuranta K.T. Miinalainen I.J. Yagi A.I. Schmitz W. Kastaniotis A.J. Airenne T.T. Gurvitz A. Hiltunen K.J. Mol. Cell. Biol. 2001; 21: 6243-6253Crossref PubMed Scopus (63) Google Scholar).With the exception of the hfa1Δ mutant, which is completely respiratory-deficient only at higher temperatures, yeast strains with lesions in the mitochondrial FAS pathway exhibit several severe mitochondrial dysfunction phenotypes when grown at 30 °C. All of the strains are respiratory-deficient, exhibit a loss of mitochondrial cytochromes, and have low levels of lipoic acid. In contrast to the small mitochondria seen in deletion mutants, overexpression of Etr1 or Htd2 results in swelling of the mitochondrial compartment (19Kastaniotis A. Autio K. Sormunen R. Hiltunen J. Mol. Microbiol. 2004; 53: 1407-1421Crossref PubMed Scopus (67) Google Scholar, 20Torkko J.M. Koivuranta K.T. Miinalainen I.J. Yagi A.I. Schmitz W. Kastaniotis A.J. Airenne T.T. Gurvitz A. Hiltunen K.J. Mol. Cell. Biol. 2001; 21: 6243-6253Crossref PubMed Scopus (63) Google Scholar).Subsequent to the yeast work, several human FAS type II homologs were isolated and characterized (Fig. 1), and most have been shown to complement the yeast mutant phenotypes (8Autio K.J. Kastaniotis A.J. Pospiech H. Miinalainen I.J. Schonauer M.S. Dieckmann C.L. Hiltunen J.K. FASEB J. 2008; 22: 569-578Crossref PubMed Scopus (44) Google Scholar, 22Zhang L. Joshi A.K. Hofmann J. Schweizer E. Smith S. J. Biol. Chem. 2005; 280: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Miinalainen I. Chen Z. Torkko J. Pirila P. Sormunen R. Bergmann U. Qin Y. Hiltunen J. J. Biol. Chem. 2003; 278: 20154-20161Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In the cases that have been reported, the mammalian genes show strong expression in tissues with a high rate of respiration.Lipoic Acid and BeyondNeither the range of fatty acids produced by the mitochondrial FAS II pathway nor their roles in cellular metabolism have been determined. In fungi, there is good experimental support for the hypothesis that the pathway is the sole source of the octanoic acid precursor required for the production of the lipoic acid cofactor essential for PDH, α-ketoglutarate dehydrogenase, and glycine cleavage system function (9Schonauer M.S. Kastaniotis A.J. Hiltunen J.K. Dieckmann C.L. Mol. Cell. Biol. 2008; 28: 6646-6657Crossref PubMed Scopus (39) Google Scholar, 24Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (131) Google Scholar, 25Jordan S.W. Cronan Jr., J.E. J. Biol. Chem. 1997; 272: 17903-17906Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 26Witkowski A. Joshi A.K. Smith S. J. Biol. Chem. 2007; 282: 14178-14185Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Witkowski et al. (26Witkowski A. Joshi A.K. Smith S. J. Biol. Chem. 2007; 282: 14178-14185Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) demonstrated carbon flow from a two-carbon precursor to lipoic acid to the apoH protein (lipoic acid acceptor) of the glycine cleavage system when bovine heart mitochondrial soluble extract was incubated with labeled acetyl-ACP and malonyl-CoA. Lipoic acid is regarded as a nutritional requirement for mammals (27Challem J.J. Med. Hypotheses. 1999; 52: 417-422Crossref PubMed Scopus (20) Google Scholar), and mammalian mitochondrial enzymes required for the attachment of the free acid to target proteins have been characterized (28Fujiwara K. Hosaka H. Matsuda M. Okamura-Ikeda K. Motokawa Y. Suzuki M. Nakagawa A. Taniguchi H. J. Mol. Biol. 2007; 371: 222-234Crossref PubMed Scopus (25) Google Scholar, 29Fujiwara K. Takeuchi S. Okamura-Ikeda K. Motokawa Y. J. Biol. Chem. 2001; 276: 28819-28823Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Thus, it was puzzling when a cDNA encoding a mitochondrial lipoic acid synthase was identified in a mammalian heart library (30Morikawa T. Yasuno R. Wada H. FEBS Lett. 2001; 498: 16-21Crossref PubMed Scopus (103) Google Scholar). Subsequently, it was shown that homozygotic inactivation of the lipoic acid synthase gene in mouse causes embryonic lethality, and embryo survival cannot be rescued by supplementing the diet of pregnant mothers with lipoic acid (31Yi X. Maeda N. Mol. Cell. Biol. 2005; 25: 8387-8392Crossref PubMed Scopus (102) Google Scholar). Thus, critical developmental processes require that lipoic acid be synthesized in vivo in mammalian mitochondria.In addition to octanoic acid, there is evidence accumulating that the FAS II pathway also synthesizes longer fatty acids. In all cases, enzymes of the mitochondrial FAS pathway (KAS/CEM1, HTD2, and MECR/ETR1) show broad substrate specificity with regard to chain length. The active sites accept substrates with short chains (C2) all the way up to C14–16 fatty acid derivatives (22Zhang L. Joshi A.K. Hofmann J. Schweizer E. Smith S. J. Biol. Chem. 2005; 280: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 32Autio K.J. Guler J.L. Kastaniotis A.J. Englund P.T. Hiltunen J.K. FEBS Lett. 2008; 582: 729-733Crossref PubMed Scopus (19) Google Scholar, 33Chen Z.J. Pudas R. Sharma S. Smart O.S. Juffer A.H. Hiltunen J.K. Wierenga R.K. Haapalainen A.M. J. Mol. Biol. 2008; 379: 830-844Crossref PubMed Scopus (34) Google Scholar). Interestingly, the recently published crystal structure of human MECR/ETR1 revealed a ligand-binding pocket deep enough to accommodate acyl groups up to 16 carbons in chain length (33Chen Z.J. Pudas R. Sharma S. Smart O.S. Juffer A.H. Hiltunen J.K. Wierenga R.K. Haapalainen A.M. J. Mol. Biol. 2008; 379: 830-844Crossref PubMed Scopus (34) Google Scholar). These observations of active-site pocket size suggest that the mitochondrial FAS pathway may produce fatty acids longer than octanoic acid. Curiously, the human CEM1 enzyme shows biphasic catalytic efficiencies, peaking with the use of C6 and C10 substrates, as does ETR1 with C8 and C12–14 substrates (22Zhang L. Joshi A.K. Hofmann J. Schweizer E. Smith S. J. Biol. Chem. 2005; 280: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 33Chen Z.J. Pudas R. Sharma S. Smart O.S. Juffer A.H. Hiltunen J.K. Wierenga R.K. Haapalainen A.M. J. Mol. Biol. 2008; 379: 830-844Crossref PubMed Scopus (34) Google Scholar). These kinetic properties may facilitate the generation and accumulation of the octanoyl-ACP required for lipoic acid production, and some ACP molecules may have the acyl chains extended.Data obtained from work with isolated Neurospora mitochondria indicate that the longest fatty acids produced by the mitochondrial FAS pathway in this fungus are myristic (C14) and hydroxymyristic acids (11Mikolajczyk S. Brody S. Eur. J. Biochem. 1990; 187: 431-437Crossref PubMed Scopus (61) Google Scholar). The Trypanosoma brucei mitochondrial FAS pathway synthesizes C16 palmitoic acid (34Stephens J.L. Lee S.H. Paul K.S. Englund P.T. J. Biol. Chem. 2007; 282: 4427-4436Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Where do these longer fatty acids end up? Interestingly, disruption of the T. brucei mitochondrial FAS pathway by the introduction of small interfering RNA against ACP resulted in cellular phospholipid composition changes (35Guler J.L. Kriegova E. Smith T.K. Lukes J. Englund P.T. Mol. Microbiol. 2008; 67: 1125-1142Crossref PubMed Scopus (51) Google Scholar). Some of the phenotypes of yeast mitochondrial FAS-deficient mutants such as loss of cytochromes and mitochondrial swelling upon overexpression of Etr1 or Htd2 cannot be explained simply by the loss or overproduction of octanoic acid/lipoic acid alone, and therefore, a physiological function for longer fatty acids produced by the mitochondrial FAS pathway or derivatives thereof must be postulated.In addition to lipoic acid, myristoyl-ACP was generated in the bovine heart mitochondrial extracts mentioned above (26Witkowski A. Joshi A.K. Smith S. J. Biol. Chem. 2007; 282: 14178-14185Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Although a number of transferases using acyl-ACPs instead of CoA esters as acyl group donors and glycerol-containing lipids as acceptors are found in plastids and prokaryotes (36Byers D.M. Gong H. Biochem. Cell Biol. 2007; 85: 649-662Crossref PubMed Scopus (149) Google Scholar), to the best of our knowledge, this type of transferase has not been identified in mammals. Therefore, perhaps with the exception of the 3-hydroxymyristyl-ACP found in Complex I of bovine heart mitochondria (37Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Mol. Cell. Proteomics. 2003; 2: 117-126Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), the destiny of other long chain acyl groups synthesized on ACP in protists, parasites, and mammals remains to be determined.Fatty Acid/Lipoic Acid Synthesis Is Required for tRNA Processing in YeastSome 15 years ago, there was an initial hint of a role for mitochondrial lipids in mitochondrial RNA metabolism. It was shown that mitochondrial tRNA processing in S. cerevisiae was perturbed in a strain that had a mutation in LIP5, which encodes lipoic acid synthase (38Sulo P. Martin N.C. J. Biol. Chem. 1993; 268: 17634-17639Abstract Full Text PDF PubMed Google Scholar). This enzyme inserts two sulfurs into octanoic acid to form lipoic acid (39Miller J.R. Busby R.W. Jordan S.W. Cheek J. Henshaw T.F. Ashley G.W. Broderick J.B. Cronan Jr., J.E. Marletta M.A. Biochemistry. 2000; 39: 15166-15178Crossref PubMed Scopus (178) Google Scholar). Mitochondrial tRNA processing requires RNase P, a ribonucleoprotein complex that processes the 5′-ends of tRNAs. In turn, the assembly of RNase P requires processing of a mitochondrial precursor RNA containing the RPM1 RNA subunit of RNase P and tRNAPro. A crucial early step in processing is the RNase P cleavage at the 5′-end of the tRNA, which releases RPM1 RNA for further 5′- and 3′-trimming steps. Fully processed RPM1 RNA assembles with the Rpm2 protein to form the active holoenzyme (40Dang Y.L. Martin N.C. J. Biol. Chem. 1993; 268: 19791-19796Abstract Full Text PDF PubMed Google Scholar).A recent screen in S. cerevisiae for mutants defective in mitochondrial RNA processing focused on identifying genes encoding previously uncharacterized enzymes involved in the 5′- and 3′-trimming of multigenic precursor RNAs (9Schonauer M.S. Kastaniotis A.J. Hiltunen J.K. Dieckmann C.L. Mol. Cell. Biol. 2008; 28: 6646-6657Crossref PubMed Scopus (39) Google Scholar). A strain with a deletion in the HTD2 gene, which encodes the dehydratase in the mitochondrial FAS pathway, was deficient in processing of the RPM1 precursor RNA, specifically at the RNase P cleavage site, which releases tRNAPro from the RPM1-tRNAPro precursor RNA (Fig. 2).FIGURE 2Metabolic fluxes and functional links associated with the mitochondrial FAS type II pathway in yeast. Solid arrows represent metabolic fluxes, and dashed arrows indicate functional links. The large arrow indicates that an unidentified product of fatty acid or lipoic acid synthesis or a derivative thereof affects the efficiency of RNase P processing of the RPM1-tRNAPro precursor RNA. Alternative sources of acetyl-CoA other than that generated by PDH (pyruvate bypass, amino acid breakdown, or transfer of extramitochondrially produced acetyl units) have been omitted for clarity. FAs, fatty acids; α-KDH, α-ketoglutarate dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)How does Htd2, a FAS enzyme, influence RNA processing? Several cases of mitochondrial proteins that have two independent functions have been reported. For example, the Neurospora tyrosyl-tRNA synthetase is required also for the excision of Group I introns in mitochondrial precursor RNAs (41Akins R.A. Lambowitz A.M. Cell. 1987; 50: 331-345Abstract Full Text PDF PubMed Scopus (190) Google Scholar). Different surfaces of the enzyme bind to tRNA and to Group I introns, respectively (42Paukstelis P.J. Chen J.H. Chase E. Lambowitz A.M. Golden B.L. Nature. 2008; 451: 94-97Crossref PubMed Scopus (69) Google Scholar). In another case, the Saccharomyces mitochondrial RNA polymerase has a separate domain that is required for protein synthesis (43Rodeheffer M.S. Shadel G.S. J. Biol. Chem. 2003; 278: 18695-18701Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To investigate whether Htd2 is also a dual-function protein, we surveyed the deletion strains affecting FAS II enzymes and found that they were all deficient in RPM1 RNA maturation. Thus, accumulation of the RPM1-tRNAPro precursor RNA was not the result of a defect in one protein directly involved in RNA processing but was due to loss of a product of the complete mitochondrial FAS pathway.How is the FAS type II biosynthetic pathway tied to tRNA processing through the biogenesis or activity of RNase P? In yeast, a product of the mitochondrial FAS pathway is required for either 1) specific maturation of RPM1 RNA and subsequent assembly of the RNase P holoenzyme or 2) enhancement of RNase P activity. A simple explanation would be that the Rpm2 protein component of RNase P undergoes a hitherto undiscovered lipoic acylation, but we have shown that this is not the case (9Schonauer M.S. Kastaniotis A.J. Hiltunen J.K. Dieckmann C.L. Mol. Cell. Biol. 2008; 28: 6646-6657Crossref PubMed Scopus (39) Google Scholar). Alternatively, a fatty acid or lipoic acid could associate non-covalently with the RNase P enzyme or with the substrate, or another protein with fatty acid/lipoic acid association could chaperone RNase P assembly or activity. Our hypothesis that a mitochondrial FAS pathway product plays a direct role in the maturation of RNase P is reinforced by the finding that these pathways are linked also in vertebrates. Although the details of this connection are different for yeast and vertebrates, the outcome may be the same. The connections in both phyla hint at a mechanism for the regulation of mitochondrial gene expression in response to cellular metabolism.Genetic Linkage between a Mitochondrial FAS Enzyme and an RNase P Subunit in VertebratesHuman mitochondrial FAS II components such as malonyl-CoA:ACP transferase (44Zhang L. Joshi A.K. Smith S. J. Biol. Chem. 2003; 278: 40067-40074Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), β-ketoacyl synthase (OXSM) (22Zhang L. Joshi A.K. Hofmann J. Schweizer E. Smith S. J. Biol. Chem. 2005; 280: 12422-12429Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and ETR1 (23Miinalainen I. Chen Z. Torkko J. Pirila P. Sormunen R. Bergmann U. Qin Y. Hiltunen J. J. Biol. Chem. 2003; 278: 20154-20161Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) were identified based on their similarity to corresponding bacterial and yeast proteins, but human homologs of fungal Htd2 or prokaryotic fabA-or fabZ-type dehydratases were not found by sequence matching (19Kastaniotis A. Autio K. Sormunen R. Hiltunen J. Mol. Microbiol. 2004; 53: 1407-1421Crossref PubMed Scopus (67) Google Scholar). Instead, cDNAs encoding the human dehydratase were identified by a functional cloning approach in which the respiratory-deficient htd2-1 yeast mutant strain was transformed with human cerebellum and kidney libraries, and transformants were selected for their ability to grow on medium containing a non-fermentable carbon source (8Autio K.J. Kastaniotis A.J. Pospiech H. Miinalainen I.J. Schonauer M.S. Dieckmann C.L. Hiltunen J.K. FASEB J. 2008; 22: 569-578Crossref PubMed Scopus (44) Google Scholar). The isolation of human HTD2 allowed the identification of a highly similar mitochondrial homolog in T. brucei, which had evaded characterization before the identity of the human enzyme was established (32Autio K.J. Guler J.L. Kastaniotis A.J. Englund P.T. Hiltunen J.K. FEBS Lett. 2008; 582: 729-733Crossref PubMed Scopus (19) Google Scholar). In addition, the recently characterized Mycobacterium tuberculosis 3-hydroxyacyl-ACP dehydratase is homologous to human HTD2 (45Sacco E. Covarrubias A.S. O'Hare H.M. Carroll P. Eynard N. Jones T.A. Parish T. Daffe M. Backbro K. Quemard A. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14628-14633Crossref PubMed Scopus (99) Google Scholar). Interestingly, all three proteins resemble the phaJ-type dehydratases involved in polyhydroxyalkanoate synthesis rather than the paradigmal E. coli fabA or fabZ dehydratases.Surprising, however, was the finding that the plasmids rescuing the htd2-1 yeast mutation contained human RPP14 cDNAs, which encode one subunit of the mammalian mitochondrial RNase P complex. Closer analysis of the cDNAs revealed an additional 3′-open reading frame encoding mitochondrial HTD2. The HTD2 gene was shown to be responsible for rescuing the yeast mutation. The RPP14-HTD2 bicistronic mRNA encodes two proteins with seemingly widely disparate roles and is expressed most abundantly in heart and liver, human tissues with robust mitochondrial function.The emergence of this unusual bicistronic arrangement in bony fish implies that the mRNA structure has been preserved for 400 million years. Almost all eukaryotic mRNAs are monocistronic because translation is initiated by the small ribosomal subunit, which scans from the cap along the 5′-untranslated region in search of the first AUG start codon (46Kozak M. J. Cell. Biochem. 2007; 102: 280-290Crossref PubMed Scopus (41) Google Scholar). How translation of the HTD2 dehydratase coding sequence is initiated 121 nucleotides 3′ of the RPP14 stop codon is a topic for future investigation. Regardless of the mechanism of translation initiation for the downstream reading frame, the fact that the physical association has been maintained over evolutionary time suggests that this arrangement is not spurious but allows co-transcriptional
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