Abstract
The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.
Highlights
Fatty acyl reductases (FARs) are monofunctional proteins of similar size and domain structure
These results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis
The first 480 amino acids, which contained both the NAD_binding_4 (PF07993) and male sterility (PF3015) domains, led to its original annotation as male sterility protein. If this N-terminal extremity is most similar to the human fatty acyl reductase 1 (HsFAR1) protein, its C-terminal 660 amino acids include the acyltransferase motif PF01553, and it is most related to the human glyceronephosphate-O-acyltransferase (Fig. 1A, HsGNPAT)
Summary
Fatty acyl reductases (FARs) are monofunctional proteins of similar size and domain structure. Coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis. Plant and insect FARs are devoid of such a hydrophobic domain and may be soluble or associated with the endoplasmic reticulum [3, 11] Following their synthesis, primary fatty alcohols are used as acyl acceptors by wax synthases to produce wax esters in the endoplasmic reticulum or to replace the acyl-chain of sn-1acyl-dihydroxyacetone phosphate by alkyl-dihydroxyacetone phosphate synthase (ADPS) to generate an ether bond linkage in the peroxisomes [5, 8]. Its coexpression with T. thermophila ADPS resulted in implementing ether lipid biosynthesis in yeast, suggesting that this FAR-like protein provides both substrates required by ADPS to initiate ether lipid biosynthesis in the peroxisomes
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