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Dihydroxyacetone Phosphate Acyltransferase is a Peripheral Membrane Protein
Author(s) -
Chilije Maxwell,
Hoffarth Elesha,
Bertolesi Gabriel,
McFarlane Sarah,
Zaremberg Vanina
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.875.4
Subject(s) - acyltransferase , dihydroxyacetone phosphate , biochemistry , biology , acyltransferases , enzyme , chemistry , microbiology and biotechnology , biosynthesis
Dihydroxyacetone phosphate acyltransferase(DHAPAT) catalyzes the initial acylation step in the synthesis of ether basedphospholipids in peroxisomes. DHAPAT adds an acyl group to dihydroxyacetonephosphate, a metabolic intermediate from glycolysis, to yield 1‐acyl‐dihydroxyacetonephosphate (1‐acyl‐DHAP). Ether lipids are abundant in the central nervoussystem and DHAPAT deficiency has been associated with many human disordersincluding cataracts and blindness. We will present results from studies using the tractable model organism Xenopuslaevis that also point to a role of DHAPAT in eye development. Analysis of DHAPAT expression by in situ hybridization indicated it is expressed in the lens epithelium and in the proliferative region of the ciliary marginal zone. Given its relevance and how little is known about DHAPAT, we have initiated biochemical studies aimed at fully characterizing this enzyme in terms of structure, function and regulation. Using a yeast heterologous expression system we have been able to overproduce functional X. laevis DHAPAT. A first purification scheme comprised of subcellular fractionation, protein solubilization with different detergents and ionic strength conditions followed by Ni‐NTA affinity chromatography has been developed. Our studies indicate that X laevis DHAPAT is a peripheral membrane protein that can associate‐dissociate from membranes. We are currently exploiting this novel feature for this kind of acyltransferases in order to setup the conditions to improve its purification with the goal of initiating structural studies. Support or Funding Information This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada to SM and VZ.