Plant Plasma Membrane P4 ATPase Figure 3. ALIS proteins co-localize with ALA1 to the plasma membrane. A GFP::ALA1 fusion was transiently expressed in tobacco epidermal cells in the presence of YFP-tagged ALIS (ALIS::YFP). (A,B) ALIS1; C,D: ALIS3; E,F: ALIS5. A,C,E: untreated cells. B,D,F: cells plasmolyzed in the presence of a concentrated solution of manitol. Arrows in the bright-field and overlay images indicate the relative positions of the plasma membrane and the cell wall in points at which they have detached (opposing arrows) or remain attached (facing arrows) during plasmolysis. The plasma membrane is indicated with a closed arrow and the cell wall with open arrows. Green: GFP fluorescence; magenta: YFP fluorescence; gray: bright field image; blue: autofluorescence. Panels on the right show an overlay image. Scale bar: 25 mm. doi:10.1371/journal.pone.0033042.g003 Previously, Gomez et al. [27] demonstrated that ALA1 complements the cold-sensitive phenotype of the Ddrs2 mutant strain [30] in the absence of a co-expressed ALIS subunit. We made several unsuccessful attempts to reproduce the complemen- tation in the same Ddrs2 mutant strain, in order to rule out discrepancies due to the yeast background. This could imply that ALA1 is not a functional homologue of yeast Drs2p. However, we noted that this strain gives rise to an elevated number of phenotypic revertants, especially when incubated at low temper- atures. Revertants could arise from induction in yeast of factors that facilitate function of recombinant ALA1. Several yeast P4 ATPases have been shown to require interaction with other proteins for full activity. For instance, plasma membrane-localized Dnf1p and Dnf2p are known to require phosphorylation by the kinases Fpk1p and Fpk2p in order to be active at their final location [31]. Another example is the interaction of Drs2p with ARF-GEF, a small GTPase involved in vesicle production in the TGN. This protein-protein interaction significantly stimulates lipid translocation by Drs2p [32]. Furthermore, Drs2p activity is dependent on the presence of a specific regulatory lipid [33]. Lack of complementation by ALA1 of the yeast P4 ATPase deletion mutant phenotypes could therefore be due to lack of an PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e33042