Background: Parasite fatty acid synthesis is an attractive drug target but complex and poorly understood. Results: We delineate the molecular activity of two pathways in Toxoplasma combining metabolomic and genetic analyses. Conclusion:The apicoplast is a significant source of fatty acids, and its products are further modified in the parasite endoplasmic reticulum. Significance: We define the metabolic host-parasite relationship with molecular resolution in intracellular parasites.

Saccharomyces cerevisiae is able to switch from fermentation to respiration (diauxic shift) with major changes in metabolic activity. This phenomenon has been previously studied on the transcriptional level. Here we present a parallel analysis of the yeast mitochondrial proteome and the corresponding transcriptional activity in cells grown on glucose (fermentation) and glycerol (respiration). A two-dimensional reference gel for this organelle proteome was established (available at www.biochem.oulu.fi/proteomics/), which contains about 800 intense spots. From 459 spots 253 individual proteins were identified, among them low abundant and hydrophobic proteins, and 37 proteins previously deemed hypothetical, with partially unknown cellular localization. After the diauxic shift, mitochondrial levels of only 18 proteins were changed (17 increased, with 1 decreased), among them proteins involved in the tricarboxylic acid cycle (Sdh1p, Sdh2p, and Sdh4p) and the respiratory chain (Cox4p, Cyb2p, and Qcr7p), proteins contributing to other respiratory pathways (Ach1p, Adh2p, Ald4p, Cat2p, Icl2p, and Pdh1p), and two proteins with unknown function (Om45p and Ybr230p). Apart from an overall increase in mitochondrial protein mass, the mitochondrial proteome remains remarkably constant, even in a major metabolic adaptation. This seemingly disagrees with results of the DNA microarray analyses, where a rather heterogenous up-or down-regulation of genes encoding mitochondrial proteins implies large changes in the proteome. We propose that the discrepancy between proteome and transcriptional regulation, apart from different translation efficiency, indicates a changed turnover rate of proteins in different physiological conditions.

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of ␣-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.Several oxidative decarboxylation reactions are carried out in prokaryotes and eukaryotes by multienzyme complexes. The function of these complexes requires the action of a sulfurcontaining cofactor, lipoic acid (6,8-thioctic acid) (1, 2). Lipoic acid is covalently attached via an amide linkage to a specific lysine residue on the surface of the conserved lipoyl domain of the E2 subunits of pyruvate dehydrogenase (PDH), 3 ␣-ketoglutarate dehydrogenase (␣-KDH), the branched chain ␣-keto acid dehydrogenase complexes, and the H protein of the glycine cleavage (GC) enzyme (3). The lipoyl moiety serves as a swinging arm that shuttles reaction intermediates between active sites within the complexes (1). Despite the well characterized function of lipoic acid as a prosthetic group, the mechanisms of its synthesis and attachment to proteins are the subject of ongoing investigations (4 -7). These reactions are best understood in Escherichia coli, which has two well defined pathways for lipoic acid synthesis and attachment: a de novo pathway and a salvage pathway (8). Octanoic acid, synthesized on the acyl carrier protein (ACP) (9), is the substrate for the de novo pathway. Lipoyl synthase (LipA) catalyzes the addition of two sulfur atoms to form lipoic acid from octanoic acid either before or after transfer to the target protein (10) by lipoyl(octanoyl)-ACP:protein transferase (LipB) (11, 12). The preferred order of these two reactions is attachment of oct...

Peroxisomes are multifunctional organelles with an important role in the generation and decomposition of reactive oxygen species (ROS). In this review, the ROS-producing enzymes, as well as the antioxidative defense system in mammalian peroxisomes, are described. In addition, various conditions leading to disturbances in peroxisomal ROS metabolism, such as abnormal peroxisomal biogenesis, hypocatalasemia, and proliferation of peroxisomes are discussed. We also review the role of mammalian peroxisomes in some physiological and pathological processes involving ROS that lead to mitochondrial abnormalities, defects in cell proliferation, and alterations in the central nervous system, alcoholic cardiomyopathy, and aging. Antioxid.

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.

␤-Oxidation is compartmentalized in mammals into both mitochondria and peroxisomes. Fatty acids with double bonds at even-numbered positions require for their degradation the auxiliary enzyme 2,4-dienoyl-CoA reductase, and at least three isoforms, two mitochondrial and one peroxisomal, exist in the rat. The Saccharomyces cerevisiae Sps19p is 34% similar to the human and rat mitochondrial reductases, and an SPS19 deleted strain was unable to utilize petroselineate (cis-C 18:1(6) ) as the sole carbon source, but remained viable on oleate (cis-C 18:1(9) ). Sps19p was purified to homogeneity from oleate-induced cells and the homodimeric enzyme (native molecular weight 69,000) converted 2,4-hexadienoyl-CoA into 3-hexenoyl-CoA in an NADPH-dependent manner and therefore contained 2,4-dienoyl-CoA reductase activity. Antibodies raised against Sps19p decorated the peroxisomal matrix of oleate-induced cells. SPS19 shares with the sporulation-specific SPS18 a common promoter region that contains an oleate response element. This element unidirectionally regulates transcription of the reductase and is sufficient for oleate induction of a promoterless CYC1-lacZ reporter gene. SPS19 is dispensable for growth and sporulation on solid acetate and oleate media, but is essential for these processes to occur on petroselineate.

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.

BackgroundPeroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma.Methods/Principal FindingsIn this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2 −/− mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane.Conclusions/SignificancePxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of “bulky” metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.