asymmetry in plant plasma membranes is still unclear, and the existence of phospholipid flippases in plant plasma membranes has not yet been shown. Takeda and Kasamo [23] tried to detect phospholipid flippase activity in the inside-out plasma membrane vesicles created by Brij 58-treatment using (oleoyl-C12-NBD)- phospholipids under various conditions, but could not find such an activity, although phosphatidylserine was concentrated in the inner leaflet. In the model plant Arabidopsis, 12 P4 ATPase genes are present [24]. Recently, we have demonstrated that two Arabi- dopsis P4 ATPases ALA2 [25] and ALA3 [26], localize to the prevacuolar compartment (PVC) and the Golgi apparatus, respectively, and require a b-subunit (ALIS protein) for exit from the endoplasmic reticulum (ER) and for transport of phospholip- ids. A third Arabidopsis P4 ATPase, ALA1, has been character- ized and shown to be able to transport lipids in yeast in the absence of a co-expressed plant b-subunit [27]. However, the subcellular localization of the protein was never investigated. In this work, we demonstrate the ALA1 localizes to the plant plasma membrane and has a strict requirement for a b-subunit to exit the ER. Results ALA1 is Retained in the ER in the Absence of an ALIS Protein Transient expression in tobacco epidermal cells has been used before to demonstrate that Arabidopsis P4 ATPases are retained in the ER in the absence of an ALIS protein [25,26]. In order to express and visualize ALA1 in tobacco, the genomic DNA fragment corresponding to this protein was cloned and placed under the control of its own promoter in a plant binary plasmid containing an in-frame fusion with Green Fluorescent Protein (GFP). However, this construct did not generate a detectable fluorescent signal when infiltrated in tobacco cells. To overcome this problem, the ALA1 genomic DNA was re-cloned into plasmids of the pMDC series [28], which contain a double 35 S promoter and allow for fusion of a GFP at each end of the protein of interest. Both the N- and the C-terminally tagged ALA1 gDNA constructs presented a clear fluorescent signal in membrane structures that resembled the ER (Figure 1A and not shown). In order to confirm the nature of these membranes, the ALA1 fusions were co- expressed with a construct containing a Yellow Fluorescent Protein (YFP) modified to include an HDEL ER retention signal at the C-terminal end (Figure 1B). Co-localization of both fluorescent proteins confirmed that ALA1 resides in the ER membrane when expressed alone. Under our experimental conditions, no bleed-through of the fluorescence signals was detected (see Figure S1). ALA1 Leaves the ER and Localizes to the Plasma Membrane in the Presence of an ALIS Protein In order to investigate whether ALA1 is an ER-resident protein or whether it leaves the ER upon interaction with an ALIS b- subunit, we transiently expressed the N-terminally GFP-tagged ALA1 fusion in tobacco leaves in the presence of untagged versions of three different ALIS proteins (ALIS1, ALIS3, ALIS5) [26]. Co-expression with any ALIS protein resulted in localization of the GFP fluorescence in membranes resembling either the plasma membrane or the tonoplast (Figure 2A,C,E). In tobacco epidermal cells, the vacuole occupies almost al the volume of the cell, pressing all other organelles against the rigid cell wall. Thus, under normal conditions, it is not possible to distinguish the plasma membrane from the tonoplast, as they both run along the Plant Plasma Membrane P4 ATPase cell wall. However, while the tonoplast can freely shrink under hyperosmotic conditions, the plasma membrane is attached to the cell wall at several contact points along the surface of the cell [29]. Thus, plasmolysis of the cells by addition of a hyperosmotic solution allows distinguishing between the two membranes. When the cells were plasmolyzed in the presence of a hyperosmotic mannitol solution, several detachment areas flanked by attachment points to the cell wall could be seen for the GFP-containing membranes, which is to be expected for a plasma membrane localization (Figure 2B,D,F). The same result was obtained for an ALA1::GFP fusion (not shown). ALIS Proteins are Localized to the Plasma Membrane When Co-expressed with ALA1 Previously we have demonstrated that the localization determi- nants for the ALA/ALIS complex reside in the a-subunit and that ALIS proteins are localized to the same compartment as the ALA protein they are co-expressed with. This was shown both for Golgi-localized ALA3 [26] and for the prevacuolar compartment- resident protein ALA2 [25]. To investigate if this was also the case for plasma membrane-localized ALA1, C-terminally YFP-tagged versions of ALIS1, ALIS3 and ALIS5 [26] were co-expressed with GFP::ALA1 (Figure 3). In all cases, the fluorescent signals co- localized to membranes resembling the plasma membrane. As above, plasma membrane localization could be confirmed by plasmolysis of the cells. Heterologous Expression of ALA1 in a Ddrs2Ddnf1Ddnf2 Yeast Mutant Strain Heterologous expression of plant proteins in the host S. cerevisiae is a common tool to obtain protein for functional analysis and to test whether gene expression in this host can functionally complement mutations in related yeast genes. For this purpose, a cDNA fragment codifying for haemagglutinin (HA)-tagged ALA1 was cloned alone or together with Arg-Gly-Ser-His6 (RGSH6) tagged ALIS1, ALIS3 or ALIS5 into yeast plasmids that contain a bi-directional GAL1-10 promoter, which allows for expression of two proteins at the same time, and introduced into a Ddrs2Ddnf1Ddnf2 yeast mutant strain. To verify expression of ALA1 in yeast, we took advantage of the epitope tags engineered at the N-terminal end of the proteins. Immunodetection in total yeast membranes with antibodies raised against the HA or the RGSH6 epitopes showed that ALA1 is not expressed to detectable levels (Figure 4A), while clear signals could be seen for HA-tagged ALA3 (used as a positive control) and for all RGSH6-tagged ALIS proteins. In order to increase the level of expression, we searched for genetic elements in the introduced cDNA that could possibly affect negatively heterologous expres- sion in yeast and identified a putative transcription termination signal located about 300 amino acid residues before the stop codon of ALA1 (see Figure S2). Using an overlapping PCR strategy, this transcription termination site was removed from the cDNA sequence. The new ALA1 version, ALA1noTT, was expressed in yeast, both in untagged or tagged versions, alone or together with RGSH6-tagged ALIS1, ALIS3 and ALIS5. Following this approach, the HA-tagged version of ALA1noTT was also not detectable in total yeast membranes (Figure 4A). Next, we used a graphical codon usage analyser to identify rare codons that might result in low levels of expression of ALA1 in S. cerevisiae. Based on the results of this analysis, five codons codifying for arginine were mutated from CGG to AGA or AGG, generating a yeast-enhanced ALA1 (yeALA1) (Figure S2). Simultaneously, we aimed at increasing expression levels by PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e33042