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J. Biol. Chem., Vol. 283, Issue 17, 11199-11209, April 25, 2008

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Membrane Topology and Essential Amino Acid Residues of Phs1, a 3-Hydroxyacyl-CoA Dehydratase Involved in Very Long-chain Fatty Acid Elongation^*

Akio Kihara¹², Hiroko Sakuraba¹, Mika Ikeda, Aki Denpoh, and Yasuyuki Igarashi

From the Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812 and the Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Advanced Life Sciences, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan

Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

Received for publication, November 1, 2007 , and in revised form, February 12, 2008.

^* This work was supported by Grant-in-aid for Young Scientists (A) 17687011 and Grant-in-aid for Scientific Research (B) 18370050 from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 81-11-706-3971; Fax: 81-11-706-4986; E-mail: kihara{at}pharm.hokudai.ac.jp.

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