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Charakterisierung der vektoriellen Acylierung als Mechanismus des Fettsäuretransports in Synechocystis sp. PCC 6803

By Simon Hans Arnfrid Sittich Freiherr von Berlepsch


Transfer of fatty acids across biological membranes is a largely uncharacterized process. In unicellular organisms, like Escherichia coli or Saccharomyces cerevisiae vectorial acylation by acyl-activating enzymes is discussed as mechanism for fatty acid transport. In the present work, studies on the only acyl-acyl-carrier protein synthetase SynAas of Synechocystis sp. PCC 6803 with regard to fatty acid transport were performed. Cells deficient in SynAas were highly resistant to externally provided α-linolenic acid. Upon treatment with external α-linolenic acid the wild type showed continuous accumulation of α-linolenic acid in cellular lipids and as free fatty acid in the cell. Long-term incubation with α-linolenic acid for 10 hours led to structural changes in the integrity of thylakoid membranes and a dramatic increase in electron transport rate (ETR) in wild type cells. After 24 hours of incubation the accumulation resulted in bleaching and dying of the wild type cells, whereas the loss of function mutant ∆synaas was completely unaffected by this treatment. Furthermore, Saccharomyces cerevisiae wild type cells displayed sensitivity against α-linolenic acid, whereas the “loss of function” mutant in the fatty acid importer FAT1p showed resistance upon α-linolenic acid incubation. Heterologous expression of SynAas in yeast wild type and ∆fat1 mutant cells resulted in an increased sensitivity against exogenous α-linolenic acid, which implies elevated levels of uptake of toxic α-linolenic acid through SynAas. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate [14C]-α-linolenic acid retrieval from preloaded liposome membranes. Taken together, the data show that the acyl-activating enzyme SynAas is necessary and sufficient to mediate transfer of fatty acids across a biological membrane. These facts were used to derive a model for α-linolenic acid uptake and metabolism in Synechocystis. In addition, the ABC- Transporter SynAbc could be identified as putative fatty acid exporter. The SynAbc “loss-of-function” mutant ∆synabc exhibited increased sensitivity upon α-linolenic acid treatment and massive accumulation of exogenously applied fatty acid, compared to wild type. The ∆synaas mutant secreted free fatty acids into the media. A simultaneous defect in the SynAbc gene completely prevented accumulation of fatty acids in the media, which gives rise to the assumption that SynAbc facilitates fatty acid export in Synechocystis

Topics: ddc:150
Year: 2012
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