Biochemical characterization of the first step in ether lipid biosynthesis: acylation of dihydroxyacetone phosphate

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dc.contributor.advisorZaremberg, Vanina
dc.contributor.authorChilije, Maxwell F. J.
dc.contributor.committeememberEdwards, Robert A.
dc.contributor.committeememberMoorhead, Greg B. G.
dc.date2020-02
dc.date.accessioned2020-01-16T16:33:07Z
dc.date.available2020-01-16T16:33:07Z
dc.date.issued2020-01
dc.description.abstractGlycerolipid 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.en_US
dc.identifier.citationChilije, 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.en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/37459
dc.identifier.urihttp://hdl.handle.net/1880/111502
dc.language.isoengen_US
dc.publisher.facultyScienceen_US
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en_US
dc.subjectLipid metabolismen_US
dc.subjectGlycerophospholipidsen_US
dc.subjectdihydroxyacetone phosphate acyltransferaseen_US
dc.subjectDHAPATen_US
dc.subjectGNPATen_US
dc.subjectGPATen_US
dc.subjectEther lipidsen_US
dc.subjectPlasmalogenen_US
dc.subjectPeroxisomeen_US
dc.subjectLipid dropletsen_US
dc.subjectYeasten_US
dc.subjectLipid binding proteinen_US
dc.subject.classificationBiology--Molecularen_US
dc.subject.classificationBiochemistryen_US
dc.titleBiochemical characterization of the first step in ether lipid biosynthesis: acylation of dihydroxyacetone phosphateen_US
dc.typedoctoral thesisen_US
thesis.degree.disciplineBiological Sciencesen_US
thesis.degree.grantorUniversity of Calgaryen_US
thesis.degree.nameDoctor of Philosophy (PhD)en_US
ucalgary.item.requestcopytrueen_US
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