Plant Plasma Membrane P4 ATPase Figure 1. ALA1 expressed without a b-subunit localizes to the ER in planta. A GFP::ALA1 fusion was transiently expressed in tobacco epidermal cells alone (A) or in the presence of an YFP::HDEL ER-retained marker (B). Green: GFP fluorescence; blue: autofluorescence; magenta: YFP fluorescence; gray: bright field image. Panels on the right show an overlay image. Scale bar: 25 mm. doi:10.1371/journal.pone.0033042.g001 modifying the GAL1 galactose-inducible promoter contained in our yeast plasmids to include the leader sequence from the strong yeast PMA1 promoter. A RGSH10 tag was also included for immunodetection of expressed proteins. A new construct containing yeALA1 under the control of the modified promoter was transformed into Ddrs2Ddnf1Ddnf2 together with plasmids either empty or containing a RGSH6-tagged ALIS1, ALIS3 or ALIS5 fusion. Following isolation of yeast microsomes, tagged proteins representing ALA1 as well as ALIS1, ALI3 and ALIS5 could now be clearly detected in protein blots (Figure 4B). In order to compare all versions of the ALA1 gene expressed from the same plasmid and bearing the same tag, homologous recombination in yeast was used to generate plasmids containing the cDNA fragments codifying for ALA1 and ALA1noTT under the control of the GAL1-10 promoter::PMA1 leader fusion. Both constructs were transformed into Ddrs2Ddnf1Ddnf2 together with plasmids either empty or containing RGSH6-tagged ALIS1, ALIS3 or ALIS5, as above. In this case, no ALA1 protein could be immunodetected by western blot (Figure 4B), indicating that, under our experimental conditions, modification of the codon usage for Arg in yeALA1 was crucial for obtaining detectable levels of expression for the protein. In previous studies, we noted that under our working conditions a single Ddrs2 yeast mutant shows a high incidence of genetic revertants when tested for growth at temperatures around 20uC, while a Ddrs2Ddnf1Ddnf2 strain is a more reliable system for testing functional complementation of the drs2 related cold-sensitive phenotype [26]. Moreover, translocation of natural lipids by heterologously expressed P4 ATPases can be tested in this triple mutant strain by growth assays on plates containing low concentrations of cytotoxic peptides that bind to endogenous phosphatidylserine (papuamide B) and phosphatidylethanolamine (duramycin) [25]. When a RGSH10:yeALA1 fusion was expressed alone in the Ddrs2Ddnf1Ddnf2 strain under the control of a galactose-inducible promoter, the plant P4 ATPase failed both to complement the cold-sensitive phenotype and to restore transport of endogenous phosphatidylserine or phosphatidyletanolamine (Figure S3). Plasma membrane localized P4 ATPases which have phosphatidylcholine as a substrate are in some cases capable of transporting the toxic choline ether lipid miltefosine to the interior of the cell [17]. Employing this assay, we were unable to demonstrate yeALA1-mediated internalization of miltefosine (Figure S3). Identical results were obtained for an untagged yeALA1. A possible explanation for the apparent inability of ALA1 to complement the phenotypes of the Ddrs2Ddnf1Ddnf2 yeast mutant strain could be a defect in targeting of the plant protein expressed in the yeast cells. In order to investigate whether lack of complementation could be due to protein mistargeting in the heterologous host, plasma membrane-enriched membrane frac- tions from yeast expressing RGSH10-tagged ALA1 alone or together with a representative member of the ALIS family (RGSH6:ALIS1) were subjected to discontinuous sucrose gradient fractionation. The results indicate that ALA1 is in fact capable of reaching the plasma membrane when overexpressed in yeast, both alone and with ALIS1 (Figure S4). Similar results have been reported previously for other members of the ALA family [25,26]. Discussion In this work we provide the first evidence for the presence of a P4 ATPase in plant plasma membranes. In animal cells, P4 ATPases have been implicated in generation of phospholipid asymmetry across the two bilayers of the membrane, a process hypothesized to be a prerequisite for endocytosis and other important functions of the plasma membrane. Expression of fluorescently-tagged versions of ALA1 in leaves of Nicotiana benthamiana proved that the protein is retained in the ER when no putative b-subunit is present. Co-expression with each of three different Arabidopsis ALIS proteins (ALIS1, ALIS3 and ALIS5) resulted in ALA1 exit from the ER and trafficking of this protein to the plasma membrane, demonstrating that this P4 ATPase is also strictly dependent on interaction with a b-subunit to reach its final subcellular localization. Moreover, when YFP-tagged versions of the ALIS proteins (normally retained in the ER; [25]) were co- expressed with GFP-tagged ALA1, both fluorescent signals could be co-localized in the plasma membrane, indicating that both proteins travel together to the destination membrane. These results are in agreement with previous studies with Golgi-localized ALA3 and prevacuolar compartment-localized ALA2, which indicate that the localization determinants for the complex formed between the P4 ATPase and its b-subunit are present in the a - catalytic subunit [25]. We further aimed at expressing ALA1 in a Ddrs2Ddnf1Ddnf2 yeast strain, which has successfully been used before for PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e33042