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Papers In Press, published online ahead of print January 1, 2004
J. Lipid Res., doi:10.1194/jlr.M300339-JLR200

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Journal of Lipid Research, Vol. 45, 32-40, January 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat 6-desaturase activity

Hervé Guillou, Sabine D'Andrea, Vincent Rioux, Romain Barnouin, Stéphanie Dalaine, Frédérique Pedrono, Sophie Jan and Philippe Legrand¹

Laboratoire de Biochimie, Institut National de la Recherche Agronomique-Ecole Nationale Supérieure Agronomique, Rennes, France

¹ To whom correspondence should be addressed. e-mail: philippe.legrand{at}agrorennes.educagri.fr

The 6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat 6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of 6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and 6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif.

These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for 6-desaturase activity.

Abbreviations: FCS, fetal calf serum; GC, gas chromatography

Supplementary key words FADS2 • polyunsaturated fatty acid biosynthesis • hexadecenoic acid

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