Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctiona l Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3,2-isomerase; ECI) are though t to be mitochondrial auxiliary enzymes involved in the beta-oxidation of u nsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signal s (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal l ocation of the two mammalian proteins can be examined in yeast, since beta- oxidation of unsaturated fatty acids is a compartmentalized process in Sacc haromyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19 p) and peroxisomal 3,2-isomerase (Eci1p). A yeast sps19 Delta mutant expres sing human 2,4-reductase ending with the native C-terminus could not grow o n petroselinic acid [cis-C-18:1(6)] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2,4-reductase but that it is prob ably not peroxisomal. Rat 3,2-isomerase expressed in a yeast eci1 Delta str ain was able to reestablish growth on oleic acid [cis-C-18:1(9)] medium irr espective of an SKL extension. Since we had shown that Delta(2,4) double bo nds could not be metabolized extra-peroxisomally to restore growth of the s ps19 Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3 ) unsaturated metabolite of oleic acid by replacing the mutant's missing ac tivity from within the peroxisomes. Immunoblotting of fractionated yeast ce lls expressing rat 3,2-isomerase in combination with electron microscopy su pported our proposal that the protein functioned in peroxisomes. The result s presented here shed new light on the function and location of human mitoc hondrial 2,4-reductase and rat monofunctional 3,2-isomerase.