Biological membranes consist of lipid bilayers. The lipid compositions between the two leaflets of the plasma membrane differ, generating lipid asymmetry. Maintenance of proper lipid asymmetry is physiologically quite important, and its collapse induces several cellular responses including apoptosis and platelet coagulation. Thus, a change in lipid asymmetry must be restored to maintain "lipid asymmetry homeostasis." However, to date no lipid asymmetry-sensing proteins or any related downstream signaling pathways have been identified. We recently demonstrated that expression of the putative yeast sphingoid long-chain base transporter/translocase Rsb1 is induced when glycerophospholipid asymmetry is altered. Using mutant screening, we determined that the pH-responsive Rim101 pathway, the protein kinase Mck1, and the transcription factor Mot3 all act in lipid asymmetry signaling, and that the Rim101 pathway was activated in response to a change in lipid asymmetry. The activated transcription factor Rim101 induces Rsb1 expression via repression of another transcription repressor, Nrg1. Changes in lipid asymmetry are accompanied by cell surface exposure of negatively charged phospholipids; we speculate that the Rim101 pathway recognizes the surface charges. INTRODUCTIONBiological membranes are composed of lipid bilayers, and in the plasma membrane the lipid compositions differ between the two leaflets, resulting in asymmetry. Glycerophospholipids and sphingolipids contribute greatly to such asymmetry. Phosphatidylcholine (PC) and complex sphingolipids, including sphingomyelin (SM) and glycosphingolipids in mammals and myo-inositol-containing sphingolipids in the yeast Saccharomyces cerevisiae, are located mainly in the outer (extracytosolic) leaflet. Conversely, phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) are confined to the inner (cytosolic) leaflet (Pomorski et al., 2004;Ikeda et al., 2006). The hydrophilic nature of the headgroups of these amphiphilic lipids hinders their ability to traverse the hydrophobic membrane interior, and spontaneous flip-flop of the protein-free model membrane occurs only in low frequency (Bai and Pagano, 1997). However, situated in the biological membranes are enzymes called lipid translocases or flippases, which catalyze the transbilayer movement of lipids and establish their asymmetry. Recent studies have identified three classes of translocases/transporters for glycerophospholipids: ABC transporters, P-type ATPases, and scramblases (Holthuis and Levine, 2005;Ikeda et al., 2006). ABC transporters catalyze flop, which is the movement from the cytosolic to extracytosolic leaflet, whereas P-type ATPases stimulate flip, the reverse movement. Scramblases randomize the lipid distribution between the two leaflets. Sometimes, discriminating between a translocase and transporter is difficult. For example, it is unclear whether a lipid in one leaflet is directly translocated to the other leaflet or is first transported to the other side of the hydrophil...

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 membranespanning 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.