A Putative Plant Aminophospholipid Flippase, the Arabidopsis P4 ATPase ALA1, Localizes to the Plasma Membrane following Association with a b-Subunit Rosa L. Lo´ pez-Marque´ s*, Lisbeth R. Poulsen, Michael G. Palmgren Department of Plant Biology and Biotechnology, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Danish National Research Foundation, Frederiksberg, Denmark Abstract Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein b-subunit. Citation: Lo´ pez-Marque´s RL, Poulsen LR, Palmgren MG (2012) A Putative Plant Aminophospholipid Flippase, the Arabidopsis P4 ATPase ALA1, Localizes to the Plasma Membrane following Association with a b-Subunit. PLoS ONE 7(4): e33042. doi:10.1371/journal.pone.0033042 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received December 20, 2011; Accepted February 9, 2012; Published April 13, 2012 Copyright: 2012 Lo´ pez-Marque´s et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Danish National Research Foundation through the PUMPKIN Center of Excellence (MGP), The Danish Council for Independent Research | Natural Sciences (FNU, project number 10-083406) (RLLM), and by The Danish Government’s Globalisation Fund (LRP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rlo@life.ku.dk Introduction The plasma membrane (PM) is the outermost barrier of a cell and is responsible for the exchange of matter and information between its exterior and the interior. First recently it has been acknowledged that the asymmetric distribution of lipids between the two leaflets of the plasma membrane is important for its proper function. In animal cells, the aminophospholipids phosphatidyl- serine and phosphatidylethanolamine (PE) are in general seques- tered in the inner leaflet of the plasma membrane, whereas sphingomyelin and glycosphingolipids are enriched in the outer leaflet [1,2]. Amongst other features, asymmetric transversal distribution of phospholipids, sphingolipids, and free sterols between the cytosolic and apoplastic (exoplasmic) leaflets is required for maintaining a low permeability to solutes [3,4]. Among lipids, phosphatidylserine in particular is observed to distribute unevenly between the two leaflets [5–7]. The phospha- tidylserine asymmetry and its regulation is considered important to cellular processes such as cell-cell recognition [8], endo- and exocytosis [1], regulation of membrane surface potential [7] and activation of membrane-bound enzymes [8,9] as indicated in mammalian and yeast cells. Regulated loss of this asymmetric lipid arrangement triggers a variety of physiological events including apoptosis [10]. In the yeast Saccharomyces cerevisiae the plasma membrane phospholipid asymmetry is influenced by ATP-dependent amino- phospholipid translocases which constitute a subfamily, P4 ATPases, in the superfamily of P-type ATPase pumps [11–13]. S. cerevisiae has five P4 ATPases, among which two are localized in the plasma membrane, namely Dnf1p and Dnf2p [14]. Loss of Dnf1p and Dnf2p leads to an increased cell surface exposure of endogenous phosphatidylserine, which is enhanced by additional removal of the Golgi-localized P4 ATPase Drs2p [15]. Concurrent with an altered phospholipid arrangement in the plasma membrane, Ddrs2Ddnf1Ddnf2 cells exhibit a defect in the uptake of the endocytic tracer FM4–64 and in the ligand-induced internalization of a-factor receptor [14]. These results point to a functional link between P-type ATPase-dependent lipid translo- cation and budding of endocytic vesicles from the plasma membrane. P4 ATPases from protozoa and animal cells have also been localized to the plasma membrane and shown to be involved in phospholipid translocation across this membrane. These include Leishmania donovanii LdMT, responsible for transporting the drug miltefosine, a toxic choline ether lipid used in treatment of the leishmaniasis disease [16,17], human ATP8B1, involved in a severe liver disease in humans [18], and mouse FetA, involved in formation of the acrosomal membrane in sperm cells [19]. In addition to being involved in phospholipid flipping [18], ATP8B1 has a structural or signalling role in formation of microvilli in intestinal cells, which appears to be independent on lipid transport across the plasma membrane [20]. In plants, much less is known on the influence of lipids on the functions of plasma membranes. It is generally assumed that a transversal lipid asymmetry exists also in plant plasma membranes [21,22], but the only analysis so far conducted on plant material concluded that phosphatidylserine is the only phospholipid asymmetrically distributed between the plasma membrane leaflets [23]. The physiological significance of the phosphatidylserine PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e33042