Untersuchungen zum Fettsäuretransport durch zelluläre und peroxisomale Membranen

Abstract

Despite intensive research and numerous publications the precise mechanism by which free fatty acids cross the plasma membrane is still controversial. Within this work we addressed the question whether acyl-CoA synthetases are involved in lipid transport in Saccharomyces cerevisiae. In our previous studies we could show that the combined deletion of the acyl-CoA synthetases FAA1 and FAA4 in YB332 leads to a fatty acid secretion phenotype which is characterized by a massive export of free fatty acids during the exponential phase and a re-import of free fatty acids during the stationary phase. In order to carry out further transport studies all additional acyl-CoA synthetases were inactivated in the background of the double mutant faa1Δfaa4Δ. Our results could show that transport through the plasma membrane can take place in the absence of any acyl-CoA synthetase activity. The direction of free fatty acid transport is reversible and can be actively regulated by the metabolic state of the cell. Obviously, a specific control mechanism initiates an active export of fatty acids upon a drastic alteration in the composition of the fatty acid pool of the cell. In contrast, shortage of carbon sources, namely a starvation signal, triggers the import of exogenous fatty acids during the stationary phase. In contrast to fatty acid transport across the plasma membrane, transport of fatty acids across the peroxisomal membrane is understood in more detail. In both yeast and plants peroxisomal ABC-transporters with an essential function in the uptake of fatty acids during β-oxidation have been identified. Despite the existence of comparable elements, the mechanism of fatty acid import in plant peroxisomes appears to differ fundamentally from that of S. cerevisiae. In this work, it could be shown, that the ABC-transporter Pat1p-Pat2p from yeast cannot be functionally replaced by the plant ABC-transporter PXA1. Only the combined expression of the plant proteins PXA1 and LACS7 resulted in successful complementation of the double mutant pat1Δfaa2Δ. Therefore, the mechanism of fatty acid import in plant peroxisomes appears to be significantly different from that of S. cerevisiae. In addition, it was possible to obtain insights on the process of fatty acid transport across the peroxisomal membrane and subsequent metabolisation by β-oxidation through the manipulation of the peroxisomal acyl-CoA pool in S. cerevisiae. The combined deletion of the acyl-CoA thioesterase TES1 and the peroxisomal acyl-CoA synthetase FAA2 in YB332 led to a distinct phenotype. This mutant did not exhibit growth in minimal medium in the presence or absence of oleic acid. In addition, a drastic reduction of the cellular acyl-CoA pool was observed. Our data support the hypothesis of a tight interaction of Tes1p and Faa2p, which in combination appear to regulate the ratio of free CoA to acyl-CoA in the peroxisomes. Interestingly, the additional deletion of the peroxisomal ABC-transporter PAT1 could partly suppress the phenotype. Thus, inhibition of the import of acyl-CoA into the peroxisomes can prevent the destabilization of the CoA/acyl-CoA-ratio. Our data indicate for the first time that a degenerated peroxisomal fatty acid metabolism is able to impact the metabolism of the entire cell. Another aspect of fatty acid metabolism was investigated in plants. Very little is known regarding the function of the ABC-transporter PXA1 in Arabidopsis thaliana during the vegetative growth phase. In this work, the phenotype of the pxa1-mutant induced by a phase of prolonged darkness was investigated. An extension of the dark phase resulted in lethality for these plants, while wild-type plants showed no symptoms. Extended dark conditions led to massive damages to the membrane system. Extensive wilting was observed despite sufficient water supply. Our studies showed that under conditions of prolonged darkness, TAG functions as a transient buffer for fatty acids that can finally be released by β-oxidation. The combination of β-oxidation and TAG-synthesis resulted in constant low levels of fatty acids in the wild-type. In the pxa1-mutant, the degradation of fatty acids via β-oxidation is impaired leading to a distinct increase in the concentration of free fatty acids. The detergent-like properties of free fatty acids resulted in severe structural damage of chloroplasts and subsequent cell death. As this phenotype can be suppressed by providing exogenous sucrose, we propose that the release of fatty acids serves as a mechanism to compensate for shortage of energy during extended darkness. It can be concluded that β-oxidation plays an essential role in energy maintenance in adult plants during a phase of prolonged darkness.