Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of β-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed

Yeast as a model to investigate the mitochondrial role in adaptation to dietary fat and calorie surplus

Several research strategies are focused towards understanding the genetic basis and molecular mechanisms that regulate uptake, synthesis, deposition, and mobilization of lipids, in the context of energy homeostasis. Because of the complexity of the problem, major input comes from the use of model systems. The aim of this work was to test the feasibility of using yeast as a model organism for studies related to dietary challenges due to high fat diet and investigate the correlation between FA metabolism and oxidative metabolism. In particular, we ask to what extent the utilization of oleic acid is dependent on mitochondrial function. We studied growth on oleic acid as a sole carbon source, and oleate stress (growth in 2 and 5% oleate) in both laboratory (BY4741 wild-type and Δsco1, Δsco2, Δtgl3, Δtgl4 mutants) and natural strains, comparing the growth phenotypes with the respiratory behaviour for each strain. We confirmed that respiratory competence is fundamental for growth on oleic acid, since the respiratory deficient mutant Δsco1 was unable to grow on oleic acid. In order to understand if the ability to use oleate as carbon source and adapt to high oleate concentrations is a general trait for the Saccharomyces cerevisiae genus, we also studied some natural strains, both diploid and haploid, identifying two meiotic derivatives of SGU90 as unable to grow in oleic acid as a sole carbon source. We investigate some aspects of mitochondrial metabolism in order to gain insights on this new finding

Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

Background: Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages 6150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl2,0.31), while the other two types of channels are slightl

The Saccharomyces cerevisiae ABC subfamily D transporter Pxa1/Pxa2p co‐imports CoASH into the peroxisome

ATP-binding cassette (ABC) subfamily D transporters are important for the uptake of fatty acids and other beta-oxidation substrates into peroxisomes. Genetic and biochemical evidence indicates that the transporters accept fatty acyl-coenzyme A that is cleaved during the transport cycle and then re-esterified in the peroxisomal lumen. However, it is not known whether free coenzyme A (CoA) is released inside or outside the peroxisome. Here we have used Saccharomyces cerevisiae and isolated peroxisomes to demonstrate that free CoA is released in the peroxisomal lumen. Thus, ABC subfamily D transporter provide an import pathway for free CoA that controls peroxisomal CoA homeostasis and tunes metabolism according to the cell’s demands

Fatty Acid Oxidation in Peroxisomes: Enzymology, Metabolic Crosstalk with Other Organelles and Peroxisomal Disorders

Peroxisomes play a central role in metabolism as exemplified by the fact that many genetic disorders in humans have been identified through the years in which there is an impairment in one or more of these peroxisomal functions, in most cases associated with severe clinical signs and symptoms. One of the key functions of peroxisomes is the β-oxidation of fatty acids which differs from the oxidation of fatty acids in mitochondria in many respects which includes the different substrate specificities of the two organelles. Whereas mitochondria are the main site of oxidation of medium-and long-chain fatty acids, peroxisomes catalyse the β-oxidation of a distinct set of fatty acids, including very-long-chain fatty acids, pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid. Peroxisomes require the functional alliance with multiple subcellular organelles to fulfil their role in metabolism. Indeed, peroxisomes require the functional interaction with lysosomes, lipid droplets and the endoplasmic reticulum, since these organelles provide the substrates oxidized in peroxisomes. On the other hand, since peroxisomes lack a citric acid cycle as well as respiratory chain, oxidation of the end-products of peroxisomal fatty acid oxidation notably acetyl-CoA, and different medium-chain acyl-CoAs, to CO2 and H2O can only occur in mitochondria. The same is true for the reoxidation of NADH back to NAD+. There is increasing evidence that these interactions between organelles are mediated by tethering proteins which bring organelles together in order to allow effective exchange of metabolites. It is the purpose of this review to describe the current state of knowledge about the role of peroxisomes in fatty acid oxidation, the transport of metabolites across the peroxisomal membrane, its functional interaction with other subcellular organelles and the disorders of peroxisomal fatty acid β-oxidation identified so far in humans