Abstract
The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[(14)C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [(14)C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.
Highlights
The transfer of fatty acids across biological membranes is a largely uncharacterized process, it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum
Because a-linolenic acid-challenged wild-type cells bleached completely after 24 h of incubation, we investigated the impact of exogenously fed a-linolenic acid on photosynthesis in wild-type and Dsynaas cells after shorter periods of time using chlorophyll fluorescence
Polyunsaturated fatty acids are part of all plant membranes, they are toxic to the cell in higher concentrations
Summary
The transfer of fatty acids across biological membranes is a largely uncharacterized process, it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided a-linolenic acid, whereas wild-type cells bleached upon this treatment. Acyl-ACP Synthetase-Mediated Fatty Acid Uptake essential for the import of long-chain fatty acids into yeast cells in addition to a functional Fat1p. Tracer experiments applying [18O]acetate or [14C]acetate to spinach (Spinacia oleracea) or pea (Pisum sativum) leaves (Pollard and Ohlrogge, 1999; Koo et al, 2004) provided initial experimental verification for a model first outlined by Shine et al (1976) This model involves thioesterasemediated cleavage of acyl-acyl carrier protein (ACP) at the inner envelope membrane, transfer of the free fatty acids to the outer envelope membrane, and acylactivating enzyme activity at the outer envelope membrane. Their study showed that isolated Arabidopsis (Arabidopsis thaliana) chloroplasts activated exogenously applied laureate very rapidly through esterification to ACP and that this reaction is dependent mostly on AAE15
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