A C. elegans model for mitochondrial fatty acid synthase II: the longevity-associated gene W09H1.5/mecr-1 encodes a 2-trans-enoyl-thioester reductase.

Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17beta-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Delta mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated specifically to curtailing mitochondrial FASII in metazoans. The availability of affected survivors will help to position C. elegans as an excellent model for future pursuits in the emerging field of mitochondrial FASII research.

Physiological function of mycobacterial mtFabD, an essential malonyl-CoA:AcpM transacylase of type 2 fatty acid synthase FASII, in yeast mct1Delta cells.

Mycobacterium tuberculosis mtFabD is an essential malonyl-CoA:AcpM transacylase and is important for vital protein-protein interactions within type 2 fatty acid synthase FASII. mtFabD contacts KasA, KasB, FabH, InhA, and possibly also HadAB, HadBC, and FabG1/MabA. Disruption of mtFabD's interactions during FASII has been proposed for drug development. Here, the gene for a mitochondrially targeted mtFabD was ectopically expressed in Saccharomyces cerevisiae mct1Delta mutant cells lacking the corresponding mitochondrial malonyl-CoA transferase Mct1p, allowing the mutants to recover their abilities to respire on glycerol and synthesize lipoic acid. Hence, mtFabD could physiologically function in an environment lacking holo-AcpM or other native interaction partners.

Caenorhabditis elegans F09E10.3 encodes a putative 3-oxoacyl-thioester reductase of mitochondrial type 2 fatty acid synthase FASII that is functional in yeast.

Caenorhabditis elegans F09E10.3 (dhs-25) was identified as encoding a 3-oxoacyl-thioester reductase, potentially of the mitochondrial type 2 fatty acid synthase (FASII) system. Mitochondrial FASII is a relatively recent discovery in metazoans, and the relevance of this process to animal physiology has not been elucidated. A good animal model to study the role of FASII is the nematode C. elegans. However, the components of nematode mitochondrial FASII have hitherto evaded positive identification. The nematode F09E10.3 protein was ectopically expressed without an additional mitochondrial targeting sequence in Saccharomyces cerevisiae mutant cells lacking the homologous mitochondrial FASII enzyme 3-oxoacyl-ACP reductase Oar1p. These yeast oar1Delta mutants are unable to respire, grow on nonfermentable carbon sources, or synthesize sufficient levels of lipoic acid. Mutant yeast cells producing a full-length mitochondrial F09E10.3 protein contained NAD(+)-dependent 3-oxoacyl-thioester reductase activity and resembled the corresponding mutant overexpressing native Oar1p for the above-mentioned phenotype characteristics. This is the first identification of a metazoan 3-oxoacyl-thioester reductase (see Note Added in Proof).

The essential mycobacterial genes, fabG1 and fabG4, encode 3-oxoacyl-thioester reductases that are functional in yeast mitochondrial fatty acid synthase type 2.

Mycobacterium tuberculosis represents a severe threat to human health worldwide. Therefore, it is important to expand our knowledge of vital mycobacterial processes, such as that effected by fatty acid synthase type 2 (FASII), as well as to uncover novel ones. Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483. However, the M. tuberculosis genome encodes four additional FabG homologs, designated FabG2-FabG5, whose functions have hitherto not been characterized in detail. Of the four candidates, FabG4/Rv0242c was recently shown to be essential for the survival of M. bovis BCG. The present work was initiated by assessing the suitability of yeast oar1Delta mutant cells lacking mitochondrial 3-oxoacyl-ACP reductase activity to act as a surrogate system for expressing FabG1/MabA directed to the mitochondria. Mutant yeast cells producing this targeted FabG1 variant were essentially wild type for all of the chronicled phenotype characteristics, including respiratory growth on glycerol medium, cytochrome assembly and lipoid acid production. This indicated that within the framework of de novo fatty acid biosynthesis in yeast mitochondria, FabG1 was able to act on shorter (C(4)) acyl substrates than was previously proposed (C(8-20)) during mycolic acid biosynthesis in M. tuberculosis. Thereafter, FabG2-FabG5 were expressed as mitochondrial proteins in the oar1Delta strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity. Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter's involvement in mycobacterial FASII remains to be explored.

A novel circuit overrides Adr1p control during expression of Saccharomyces cerevisiae 2-trans-enoyl-ACP reductase Etr1p of mitochondrial type 2 fatty acid synthase.

The significance of the chronicled role of the yeast transcription factor Adr1p in regulating ETR1 was examined in wild type and isogenic adr1Delta mutant cells. An ETR1-lacZ reporter construct was used to verify Adr1p-dependent gene expression. On solid glycerol medium containing X-gal, wild-type cells expressing the reporter turned blue, whereas the adr1Delta mutants remained white. beta-Galactosidase activity measurements following 24-h cell growth in liquid glycerol medium revealed a 6.5-fold greater expression level of the reporter gene in the wild type compared with the adr1Delta mutant. In contrast, immunoblotting showed that Etr1p abundance was essentially indistinguishable between the two strains whereas Cta1p, whose expression depends on Adr1p, was present in the wild-type cells, but not in the mutants. Moreover, enzyme assays conducted on transformed wild-type and adr1Delta mutant cells expressing a plasmid-borne ETR1 tethered behind the native promoter revealed similar levels of reductase activity, and the lipoic acid content in the two parental strains was equivalent. Hence, while Adr1p influenced the transcription levels of ETR1, it did not alter the abundance of Etr1p, the level of reductase activity, or the cellular amount of lipoic acid. The results point toward a potentially novel layer of control for maintaining physiological levels of lipoic acid.

Avoiding unscheduled transcription in shared promoters: Saccharomyces cerevisiae Sum1p represses the divergent gene pair SPS18-SPS19 through a midsporulation element (MSE).

The sporulation-specific gene SPS18 shares a common promoter region with the oleic acid-inducible gene SPS19. Both genes are transcribed in sporulating diploid cells, albeit unevenly in favour of SPS18, whereas in haploid cells grown on fatty acids only SPS19 is highly activated. Here, SPS19 oleate-response element (ORE) conferred activation on a basal CYC1-lacZ reporter gene equally in both orientations, but promoter analysis using SPS18-lacZ reporter constructs with deletions identified a repressing fragment containing a midsporulation element (MSE) that could be involved in imposing directionality towards SPS19 in oleic acid-induced cells. In sporulating diploids, MSEs recruit the Ndt80p transcription factor for activation, whereas under vegetative conditions, certain MSEs are targeted by the Sum1p repressor in association with Hst1p and Rfm1p. Quantitative real-time PCR demonstrated that in haploid sum1Delta, hst1Delta, or rfm1Delta cells, oleic acid-dependent expression of SPS18 was higher compared with the situation in wild-type cells, but in the sum1Delta mutant, this effect was diminished in the absence of Oaf1p or Pip2p. We conclude that SPS18 MSE is a functional element repressing the expression of both SPS18 and SPS19, and is a component of a stricture mechanism shielding SPS18 from the dramatic increase in ORE-dependent transcription of SPS19 in oleic acid-grown cells.

Heterologous expression of mycobacterial proteins in Saccharomyces cerevisiae reveals two physiologically functional 3-hydroxyacyl-thioester dehydratases, HtdX and HtdY, in addition to HadABC and HtdZ.

We report on Mycobacterium tuberculosis Rv0241c and Rv3389c, representing two physiologically functional 3-hydroxyacyl-thioester dehydratases (Htd). These enzymes are potentially entrained in type 2 fatty acid synthase (FASII). Mycobacterial FASII is involved in the synthesis of mycolic acids, which are the major constituents of the protective layer around the pathogen, shielding it from noxious chemicals and the host's immune system. Mycolic acids are additionally associated with the virulence and resilience of M. tuberculosis. Here, Rv0241c and Rv3389c, which are distinct from the previously identified heterodimers Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC) but also the homodimer Rv0130 (HtdZ), were identified by expressing the corresponding candidate open reading frames in Saccharomyces cerevisiae htd2Delta cells lacking mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase activity, followed by scoring for phenotype rescue. The htd2Delta mutant fails to produce sufficient levels of lipoic acid and does not respire or grow on nonfermentable carbon sources. Soluble protein extracts made from mutant htd2Delta cells expressing mitochondrially targeted Rv0241c or Rv3389c contained 3-hydroxyacyl-thioester hydratase activity. Moreover, mutant yeast cells expressing Rv0241c or Rv3389c were able to recover their respiratory growth on glycerol medium and efficiently reduce 2,3,5-triphenyltetrazolium chloride. Additionally, expression of mitochondrial Rv0241c or Rv3389c in htd2Delta cells also restored de novo lipoic acid synthesis to 92 and 40% of the level in the wild-type strain, respectively. We propose naming Rv0241c and Rv3389c as HtdX and HtdY, respectively, and discuss the implications of our finding with reference to Rv0098, a candidate mycobacterial FabZ homologue with intrinsic thioesterase and hydratase activities that lacks the eukaryotic-like hydratase-2 motif.

Identification of the Leishmania major proteins LmjF07.0430, LmjF07.0440, and LmjF27.2440 as components of fatty acid synthase II.

Leishmania major causes leishmaniasis and is grouped within the Trypanosomatidae family, which also includes the etiologic agent for African sleeping sickness, Trypanosoma brucei. Previous studies on T. brucei showed that acyl carrier protein (ACP) of mitochondrial fatty acid synthase type 2 (FASII) plays a crucial role in parasite survival. Additionally, 3-oxoacyl-ACP synthase TbKASIII as well as TbHTD2 representing 3-hydroxyacyl-ACP dehydratase were also identified; however, 3-oxoacyl-ACP reductase TbKAR1 has hitherto evaded positive identification. Here, potential Leishmania FASII components LmjF07.0440 and LmjF07.0430 were revealed as 3-hydroxyacyl-ACP dehydratases LmHTD2-1 and LmHTD2-2, respectively, whereas LmjF27.2440 was identified as LmKAR1. These Leishmania proteins were ectopically expressed in Saccharomyces cerevisiae htd2Delta or oar1Delta respiratory deficient cells lacking the corresponding mitochondrial FASII enzymes Htd2p and Oar1p. Yeast mutants producing mitochondrially targeted versions of the parasite proteins resembled the self-complemented cells for respiratory growth. This is the first identification of a FASII-like 3-oxoacyl-ACP reductase from a kinetoplastid parasite.

Function of heterologous Mycobacterium tuberculosis InhA, a type 2 fatty acid synthase enzyme involved in extending C20 fatty acids to C60-to-C90 mycolic acids, during de novo lipoic acid synthesis in Saccharomyces cerevisiae.

We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C(20) fatty acids to form C(60)-to-C(90) mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Delta cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Delta cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C(4) to C(8)) than was previously thought (>C(12)). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

Identification of a novel mycobacterial 3-hydroxyacyl-thioester dehydratase, HtdZ (Rv0130), by functional complementation in yeast.

We report on the identification of Mycobacterium tuberculosis HtdZ (Rv0130), representing a novel 3-hydroxyacyl-thioester dehydratase. HtdZ was picked up by the functional complementation of Saccharomyces cerevisiae htd2Delta cells lacking the dehydratase of mitochondrial type II fatty acid synthase. Mutant cells expressing HtdZ contained dehydratase activity, recovered their respiratory ability, and partially restored de novo lipoic acid synthesis.

The biochemistry of oleate induction: transcriptional upregulation and peroxisome proliferation.

Unicellular organisms such as yeast constantly monitor their environment and respond to nutritional cues. Rapid adaptation to ambient changes may include modification and degradation of proteins; alterations in mRNA stability; and differential rates of translation. However, for a more prolonged response, changes are initiated in the expression of genes involved in the utilization of energy sources whose availability constantly fluctuates. For example, in the presence of oleic acid as a sole carbon source, yeast cells induce the expression of a discrete set of enzymes for fatty acid beta-oxidation as well as proteins involved in the expansion of the peroxisomal compartment containing this process. In this review chapter, we discuss the factors regulating oleate induction in Saccharomyces cerevisiae, and we also deal with peroxisome proliferation in other organisms, briefly mentioning fatty acid-independent signals that can trigger this process.

Modified low-density lipoproteins and high-density lipoproteins. From investigation tools to real in vivo players.

It has long been known that the oxidative state of the various plasma lipoproteins modulates platelet aggregability, thereby contributing to atherogenesis. Low-density lipoprotein (LDL), occurring in vivo both in the native and oxidised forms, interacts directly with platelets, by binding to specific receptors. While the identity of the receptors for native LDL and some subfractions of high-density lipoproteins (HDL) remains disputed, apoE-containing HDL(2) binds to LRP8. The nature of these interactions as well as the distinction between candidate receptor proteins was elucidated using covalently modified apolipoproteins, which pointed to the participation of apolipoproteins in high affinity binding. However, the platelet effects initiated by binding of native lipoproteins remain controversial. Some of this ambiguity can be traced to the fact that native LDL inevitably undergoes substantial oxidisation upon modification, including by radiolabelling. The platelet-activating effects provoked by oxidised LDL are irrefutable, but many details remain unknown. The role of CD36 in platelet binding by oxidised LDL is well established, although additional receptors may exist. Much less is known about the interaction of oxidised HDL with platelets, since platelet activation was observed in some, but not all studies. Various frequently applied in vitro oxidation methods produce modified lipoprotein species that may not be relevant in vivo. Based on the reported modifications obtained by in vitro oxidation of LDL, early investigations focused mainly on the formation and the eventual effects of oxidised lipids. More recently, alterations to lipoproteins performed using hypochloric acid and myeloperoxidase redirected the attention to the role of modified apoproteins in triggering platelet responses.

Candida tropicalis expresses two mitochondrial 2-enoyl thioester reductases that are able to form both homodimers and heterodimers.

Here we report on the cloning of a Candida tropicalis gene, ETR2, that is closely related to ETR1. Both genes encode enzymatically active 2-enoyl thioester reductases involved in mitochondrial synthesis of fatty acids (fatty acid synthesis type II) and respiratory competence. The 5'- and 3'-flanking (coding) regions of ETR2 and ETR1 are about 90% (97%) identical, indicating that the genes have evolved via gene duplication. The gene products differ in three amino acid residues: Ile67 (Val), Ala92 (Thr), and Lys251 (Arg) in Etr2p (Etr1p). Quantitative PCR analysis and reverse transcriptase-PCR indicated that both genes were expressed about equally in fermenting and ETR1 predominantly respiring yeast cells. Like the situation with ETR1, expression of ETR2 in respiration-deficient Saccharomyces cerevisiae mutant cells devoid of Ybr026p/Etr1p was able to restore growth on glycerol. Triclosan that is used as an antibacterial agent against fatty acid synthesis type II 2-enoyl thioester reductases inhibited growth of FabI overexpressing mutant yeast cells but was not able to inhibit respiratory growth of the ETR2- or ETR1-complemented mutant yeast cells. Resolving of crystal structures obtained via Etr2p and Etr1p co-crystallization indicated that all possible dimer variants occur in the same asymmetric unit, suggesting that similar dimer formation also takes place in vivo.

Saccharomyces cerevisiae PIP2 mediating oleic acid induction and peroxisome proliferation is regulated by Adr1p and Pip2p-Oaf1p.

Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively. The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE. Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2. Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type. In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type. Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells. However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control. This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid. Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.

Saccharomyces cerevisiae Pip2p-Oaf1p regulates PEX25 transcription through an adenine-less ORE.

The role of the Saccharomyces cerevisiae Pip2p-Oaf1p transcription factor was examined in reference to the regulation of the peroxin gene PEX25 involved in peroxisome proliferation. The PEX25 promoter contains an oleate response element (ORE)-like sequence comprising a CGG palindrome lacking a canonical adenine, which is considered critical for element function and Pip2p-Oaf1p binding. Pex25p levels were higher in wild-type cells grown on oleic acid medium than in those grown on ethanol, but this induction was abolished in cells devoid of Pip2p-Oaf1p. Studies based on lacZ reporter genes and in vitro protein-DNA interactions revealed that the PEX25 ORE could bind Pip2p-Oaf1p and confer activation on a basal promoter. These findings reinforced the central role played by Pip2p-Oaf1p in regulating peroxisome proliferation. We also investigated whether Pip2p-Oaf1p is important for regulating genes encoding peroxins involved in protein import into the peroxisomal matrix. Pip2p-Oaf1p was able to bind efficiently to the PEX5 ORE but not to an ORE-like CGG palindrome in the PEX14 promoter. However, immunoblotting revealed that both Pex5p and Pex14p (as well as Pex7p and Pex13p) were not more abundant in cells grown on oleic acid medium compared with ethanol. These data on a functional, adenine-less, PEX25 ORE and a nonfunctional N13-spaced ORE-like sequence in the PEX14 promoter capable of binding Pip2p-Oaf1p prompts readjustment of the ORE consensus to comprise CGGN3TNA/(R)N8-12CCG.

The biochemistry of peroxisomal beta-oxidation in the yeast Saccharomyces cerevisiae.

Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of beta-oxidation enzyme activities as well as a set of auxiliary activities to provide the beta-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of beta-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p-Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in beta-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in beta-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

The peroxisomal transporter gene ANT1 is regulated by a deviant oleate response element (ORE): characterization of the signal for fatty acid induction.

Saccharomyces cerevisiae ANT1/YPR128c encodes the peroxisomal adenine nucleotide transporter that provides ATP for intra-peroxisomal activation of medium-chain fatty acids. A lacZ reporter construct comprising the ANT1 promoter was shown to be comparatively more highly expressed in a wild-type strain grown on oleic acid, a long-chain fatty acid, than in pip2Delta(oaf1)Delta mutant cells that are defective in fatty acid induction. The ANT1 promoter was demonstrated to contain a deviant oleate response element (ORE) that could bind the Pip2p-Oaf1p transcription factor and confer activation on a basal CYC1-lacZ reporter gene. Expression of Ant1p as well as other enzymes whose genes are known to be regulated by a canonical ORE was found to be increased in cells grown on lauric acid, a medium-chain fatty acid. We concluded that the signal for induction does not differentiate between long- and medium-chain fatty acids. This signal was independent of beta-oxidation or the biogenesis of the peroxisomal compartment where this process occurs, since a pox1Delta strain blocked in the first and rate-limiting step of beta-oxidation as well as various pex mutant cells devoid of intact peroxisomes produced sufficient amounts of Pip2p-Oaf1p for binding OREs in vitro and for expressing an ORE-driven reporter gene. The signal's durability was shown to be related to the concentration of fatty acids in the medium, since a pex6Delta strain expressed an ORE-driven reporter gene at high levels for a longer period than did isogenic wild-type cells. Generation of the signal was also independent of protein synthesis, as demonstrated by cycloheximide treatment.

Targeting of malate synthase 1 to the peroxisomes of Saccharomyces cerevisiae cells depends on growth on oleic acid medium.

The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid beta-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on different carbon sources. Immunoelectron microscopy in combination with cell fractionation showed that Mls1p was abundant in the peroxisomes of cells grown on oleic acid, whereas in ethanol-grown cells Mls1p was primarily cytosolic. This was reinforced using a green fluorescent protein (GFP)-Mls1p reporter, which entered peroxisomes solely in cells grown under oleic acid-medium conditions. Although growth of cells devoid of Mls1p on ethanol or acetate could be fully restored using a cytosolic Mls1p devoid of SKL, this construct could only partially alleviate the requirement for native Mls1p in cells grown on oleic acid. The combined results indicated that Mls1p remained in the cytosol of cells grown on ethanol, and that targeting of Mls1p to the peroxisomes was advantageous to cells grown on oleic acid as a sole carbon source.

Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster.

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.