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.

Fatty acids are essential for membrane biosynthesis in all organisms and serve as signaling molecules in many animals. Here, we found that saturated very-long-chain fatty acids (VLCFAs; C20:0 to C30:0) exogenously applied in ovule culture medium significantly promoted cotton (Gossypium hirsutum) fiber cell elongation, whereas acetochlor (2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide; ACE), which inhibits VLCFA biosynthesis, abolished fiber growth. This inhibition was overcome by lignoceric acid (C24:0). Elongating fibers contained significantly higher amounts of VLCFAs than those of wild-type or fuzzless-lintless mutant ovules. Ethylene nullified inhibition by ACE, whereas C24:0 was inactive in the presence of the ethylene biosynthesis inhibitor (l-[2-aminoethoxyvinyl]-glycine), indicating that VLCFAs may act upstream of ethylene. C24:0 induced a rapid and significant increase in ACO (for 1-aminocyclopropane-1-carboxylic acid oxidase) transcript levels that resulted in substantial ethylene production. C24:0 also promoted Ser palmitoyltransferase expression at a later stage, resulting in increased sphingolipid biosynthesis. Application of C24:0 not only stimulated Arabidopsis thaliana root cell growth but also complemented the cut1 phenotype. Transgenic expression of Gh KCS13/CER6, encoding the cotton 3-ketoacyl-CoA synthase, in the cut1 background produced similar results. Promotion of Arabidopsis stem elongation was accompanied by increased ACO transcript levels. Thus, VLCFAs may be involved in maximizing the extensibility of cotton fibers and multiple Arabidopsis cell types, possibly by activating ethylene biosynthesis.

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.

Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.

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.