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• W1555676010 abstract "Fatty acids are essential components of living organism. The distribution of fatty acids between cells as well as within a cell is a complex process due to their low solubility in water. In particular the transport of fatty acids across membranes is still a focus of research. However, the mechanism underlying the transport of fatty acids across membranes is largely obscure in higher plants. In the present work, we investigated the transport of fatty acids into peroxisomes. The transport of fatty acids through the peroxisomal membrane is required for the degradation of storage lipids during germination of oilseeds. In Arabidopsis thaliana, the peroxisomal ATP-binding-cassette (ABC) transporter PXA1 was shown to be involved in this process. It was postulated that the plant PXA1 protein might perform the same function as its Saccharomyces cerevisiae homolog, namely transporting acyl-CoA molecules across the peroxisomal membrane. On the other hand the plant peroxisomal acyl-CoA synthetases LACS6 and LACS7 have been shown to be essential for further metabolization of fatty acids via s-oxidation. The contradiction between transport of activated fatty acids via PXA1 and the requirement of a peroxisomal acyl-CoA synthetase activity represented the starting point of this work. The comparison of the generated triple mutant lacs6 lacs7 pxa and the parent lines regarding the capability to degrade the triacylglycerol specific eicosenoic acid (20:1) during post-germinative growth revealed similar rates of degradation in all mutants lines examined. This result indicates that PXA1 acts in series with peroxisomal LACS. The delayed degradation of triacylglycerols in the mutant lines has to be due to a s-oxidation-independent pathway. Further analyses excluded α-oxidation as an alternative degradation process. Measurements of the acyl-CoA pool in wild type and mutant seedlings showed an increased level of very long chain acyl-CoAs in the mutants. We assume that the delayed degradation of storage lipids results from enhanced elongation of long-chain fatty acids to very-long-chain fatty acids (VLCFA). The synthesized VLCFAs could be incorporated into complex lipids like waxes or sphingolipids. To evaluate the contribution of certain LACS genes to the progress of fatty acid degradation the RNA expression profiles of all nine LACS genes during germination and postgerminative growth were examined by semi-quantitative RT-PCR. The peroxisomal LACS are already expressed during germination. Together with the onset of storage lipid metabolism also cytoplasmic LACS transcripts are detectable. Since an activation of free fatty acids in the cytoplasm is therefore possible, the substrate of the PXA1 transport protein could be both free fatty acids as well as activated fatty acids. In addition, promoter activities of PXA1 and LACS7 were localized in a variety of tissues during different developmental stages showing a very similar pattern of activity. The observed small but distinct differences in promoter activity of PXA1 and LACS7 suggest that both proteins may function also in independent pathways. These promoter analyses underline the important function of PXA1 and LACS7 beyond seedling development. The analyzed germination capacity from different PXA1-mutants showed that the potential to break dormancy in the presence of sugar is independent from the position of the mutation within the gene but is rather influenced by factors affecting seed coat integrity. For transport assays of fatty acids into peroxisomes we used intact glyoxysomes from Ricinus communis to determine the substrate specificity of PXA1. The measurements indicated that free as well as activated fatty acids were taken up by glyoxysomes without a clear preference for either one of these two substrates. For further characterization of the PXA1-transporter we tested methods for its reconstitution into liposomes based on phospholipids. Therefore PXA1 was expressed in Saccharomyces cerevisiae followed by the isolation of microsomal membranes. The integral membrane proteins were solubilized using detergent and reconstituted into lipid vesicles. With this experimental setup so far no PXA1-dependent transport was measurable. In preliminary tests we found that the membrane of liposomes devoid of proteins is impermeable for LCFA-CoA esters. On the other hand the strong influx of free fatty acids into liposomes or at least binding of free fatty acids to the membrane itself is completely unspecific. This effect could be prevented by providing the free fatty acids bound to BSA. Data of further experiments supported the idea that vesicles trapped with an acyl-CoA synthetase facilitate long chain fatty acid permeation. According to this data no protein is necessary for the translocation of free fatty acids across the membrane. Furthermore, we suggest that a direct protein-protein interaction between PXA1 and peroxisomal LACS is not required for efficient fatty acid degradation. Saccharomyces cerevisiae contains two homologous sequences to PXA1 encoded by PAT1 and PAT2. In addition two potential acyl-CoA-synthetases are localized to peroxisomes, Faa2p and Fat2p. However previous attempts to demonstrate enzymatic activity of Fat2p failed. Growth analyses were performed to complement the pat1Δ growth phenotype on medium containing oleic acid. Therefore we generated pat1Δ and pat1Δfaa2Δ mutant strains and transformed them with plasmids encoding PXA1 of Arabidopsis. The results obtained indicated that PXA1 were not able to functionally complement PAT1. Further investigations showed that a deletion of FAT2 in combination with deletion of either PAT1 or FAA2 respectively, is synthetically lethal for growth on media containing oleic acid. Because of these phenotypes and first activity tests we consider the possibility that Fat2p is an acyl-CoA synthetase with at least low activity that was disregarded in the present import model. We postulate in opposite to the current perception a possible role for a peroxisomal LACS activity in yeast similar to the situation in plants." @default.
• W1555676010 created "2016-06-24" @default.
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• W1555676010 date "2007-08-15" @default.
• W1555676010 modified "2023-09-26" @default.
• W1555676010 title "Fettsäuretransport in die peroxisomale Matrix von Arabidopsis thaliana" @default.
• W1555676010 hasPublicationYear "2007" @default.
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