Plant Plasma Membrane P4 ATPase Figure 2. ALA1 localizes to the plasma membrane in the presence of an ALIS protein. A GFP::ALA1 fusion was transiently expressed in tobacco epidermal cells in the presence of an untagged ALIS. (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; blue: autofluorescence; gray: bright field image. Panels on the right show an overlay image. Scale bar: 25 mm. doi:10.1371/journal.pone.0033042.g002 characterization of lipid translocation by the plant P4 ATPases ALA2 and ALA3 [25,26]. After optimization of the expression levels for ALA1 in yeast, we were successful in expressing recombinant protein in amounts that could readily be detected in yeast extracts by immunological methods. Following expression of ALA1 alone and together with ALIS genes we were not able to demonstrate functional complementation of the cold-sensitive Drs2p-related phenotype or to recover the loss of phospholipid asymmetry generated by deletion of the endogenous P4 ATPases in the mutant yeast strain. This lack of complementation was not derived from a defect in targeting of ALA1 within the yeast trafficking machinery, as the plant P4 ATPase overexpressed in yeast was found to reach the yeast plasma membrane both in the presence or the absence of an ALIS b-subunit. We could not detect any ATPase activity above background levels in membranes from transformed yeast cells and also failed to demonstrate the formation of an acyl-phosphate bond in the recombinant protein (data not shown). The inability of ALA1 expression to complement the phenotypes of the mutant yeast strain could therefore be due to lack of functionality of the recombinant protein, for example if an unknown factor (e.g. protein or lipid) present in plants but not in yeast is required for ALA1 activity. PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e33042