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J. Biol. Chem., Vol. 284, Issue 2, 954-965, January 9, 2009

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Lipid-engineered Escherichia coli Membranes Reveal Critical Lipid Headgroup Size for Protein Function^*

Malin Wikström, Amélie A. Kelly, Alexander Georgiev, Hanna M. Eriksson, Maria Rosén Klement, Mikhail Bogdanov, William Dowhan¹, and Åke Wieslander²

From the Department of Biochemistry and Biophysics, Stockholm University, Stockholm 10691, Sweden and the Department of Biochemistry and Molecular Biology, University of Texas School of Medicine, Houston, Texas 77225

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.

Received for publication, June 12, 2008 , and in revised form, October 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R37 GM20478 (to W. D.). This work was also supported by the Swedish Research Council and the Wenner-Gren Foundations (to Å. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. Tel.: 713-500-6051; Fax: 713-500-0652; E-mail: William.Dowhan{at}uth.tmc.edu. ² To whom correspondence may be addressed. Tel.: 46-8-162463; Fax: 46-8-153679; E-mail: ake{at}dbb.su.se.

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