Abstract
Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.
Highlights
Which replaces the acyl chain of 1-acyl-DHAP with a long chain fatty alcohol [1, 3]
fatty acyl-CoA reductase 1 (Far1) Is Essential for Plasmalogen Synthesis—Ether-linked alkyl bond formation in ether lipids is catalyzed by alkyl dihydroxyacetone phosphate synthase (ADAPS) and requires fatty alcohols that are converted from fatty acids by reduction of fatty acyl-CoAs
Two types of Fatty acyl-CoA reductase (Far) cDNAs, FAR1 and FAR2, were cloned from human and mouse, and their peroxisomal localization was demonstrated by expressing epitope-tagged Far proteins [8]
Summary
Lipid Analysis—Cells were metabolically labeled with [14C]palmitate as described in the supplemental Experimental Procedures. Lipids were extracted from cells by the Bligh and Dyer method [9], dissolved in 0.5 M sodium methoxide in anhydrous methanol, and incubated at 50 °C for 20 min. After neutralization with acetic acid, lipids were re-extracted by the Bligh and Dyer method [9]. To detect plasmenylethanolamine (PlsEtn) by one-dimensional TLC, PlsEtn was converted to either 2-acyl-glycerophosphoethanolamine (GPE) by trichloroacetic acid treatment [3] or 1-alkenylGPE by alkaline methanolysis. SiRNA-mediated Knockdown of FAR1—FAR1 knockdown in HeLa cells was performed using StealthTM siRNA. The primers used were: human FAR1 siRNA (target sequence, 5Ј-CCACTTTCAAGAGGAATCCTCTCGA-3Ј), human FAR1 siRNA (target sequence, 5Ј-GAGATGCTGTTCAGTTAAATGTGAT-3Ј), human FAR1 siRNA
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