Biochemical characterization of the first step in ether lipid biosynthesis: acylation of dihydroxyacetone phosphate
Date
2020-01
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Abstract
Glycerolipid biosynthesis is essential for cellular growth and proliferation. It is an indispensable pathway in many living organisms. In mammals four GPAT isoforms are known to catalyze the first step in glycerolipid biosynthesis which involves acylation of glycerol-3-phosphate to lysophosphatidic acid. GPATs 1 and 2 localize to the mitochondria while GPATs 3 and 4 localize to the ER. This redundancy is poorly understood. A fifth acyltransferase, DHAPAT, known to metabolize dihydroxyacetone phosphate, resides in peroxisomes. The principal function of DHAPAT in mammals is the biosynthesis of ether-based lipids which are widely distributed in the body and noticeably abundant in membranes of the nervous system. Compared to the four GPATs, DHAPAT is understudied. Current knowledge associating DHAPAT deficiency with many human disorders has re-kindled the interest in the research community. Symptoms of DHAPAT deficiency have been observed in many peroxisomal disorders including Zellweger syndrome and Rhizomelic Chondrodysplasia Punctata type 2. Understanding of this enzyme together with the roles played by ether lipids, could help find ways of alleviating some of the problems associated with these disorders. In the current study, we have performed a biochemical characterization of animal DHAPATs using heterologous expression in the yeast Saccharomyces cerevisiae. We confirmed that DHAPAT is a peripheral membrane protein. Interestingly, DHAP acylation alone was sufficient to support life of a yeast strain devoid of endogenous GPATs (sct1Δ gpt2Δ). This phenotype was therefore used as part of a functional in vivo assay to identify regions of the enzyme required for membrane binding. These investigations unveiled a critical role of the amino end of the protein in mediating the association of DHAPAT to membranes. Surprisingly, in vivo imaging of yeast expressing animal DHAPATs revealed association of the enzyme with LDs. This feature was also conserved in the human DHAPAT overexpressed in HeLa cells. Our findings support a dual localization of DHAPAT in peroxisomes and LDs. Therefore, in addition to the well accepted peroxisomal targeting of DHAPAT, a conserved LD targeting mechanism also exists, mediated by the amino terminus of the protein.
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Keywords
Lipid metabolism, Glycerophospholipids, dihydroxyacetone phosphate acyltransferase, DHAPAT, GNPAT, GPAT, Ether lipids, Plasmalogen, Peroxisome, Lipid droplets, Yeast, Lipid binding protein
Citation
Chilije, M. F. J. (2020). Biochemical characterization of the first step in ether lipid biosynthesis: acylation of dihydroxyacetone phosphate (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.