The Putative Aminophospholipid Translocases, DNF1 and DNF2, Are Not Required for 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-phosphatidylserine Flip across the Plasma Membrane of Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry 2008-12, Vol.283 (50), p.35060-35069
• Main Authors: Stevens, Haley C., Malone, Lynn, Nichols, J. Wylie
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - chemistry; Alleles; ATP-Binding Cassette Transporters - metabolism; Cell Membrane - metabolism; DNA-Binding Proteins - metabolism; Flow Cytometry; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Lipids and Lipoproteins: Metabolism, Regulation, and Signaling; Phosphatidylserines - chemistry; Phosphatidylserines - pharmacology; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - metabolism; Temperature; Trans-Activators - metabolism; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M802379200

The regulation of phosphatidylserine (PS) distribution across the plasma membrane of eukaryotic cells has been implicated in numerous cell functions (e.g. apoptosis and coagulation). In a recent study, fluorescent phospholipids labeled in the acyl chain with 7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD) were used to identify two members of the P4 subfamily of P-type ATPases, Dnf1p and Dnf2p, that are necessary for the inward-directed transport of phospholipids across the plasma membrane (flip) of yeast (Pomorski, T., Lombardi, R., Riezman, H., Devaux, P. F., Van Meer, G., and Holthuis, J. C. (2003) Mol. Biol. Cell14, 1240-1254). Herein, we present evidence that the flip of NBD-labeled PS (NBD-PS) across the plasma membrane does not require the expression of Dnf1p or Dnf2p. In strains in which DNF1 and DNF2 are both deleted, the flip of NBD-PS is increased ∼2-fold over that of the isogenic parent strain, whereas the flip of NBD-labeled phosphatidylcholine and NBD-labeled phosphatidylethanolamine are reduced to ∼20 and ∼50%, respectively. The mechanism responsible for NBD-PS flip is similar to that for NBD-labeled phosphatidylcholine and NBD-labeled phosphatidylethanolamine in its dependence on cellular ATP and the plasma membrane proton electrochemical gradient, as well as its regulation by the transcription factors Pdr1p and Pdr3p. Based on the observation that deletion or inactivation of all four members of the DRS2/DNF essential subfamily of P-type ATPases does not affect NBD-PS flip, we conclude that the activity reflected by NBD-PS internalization is not the essential function of the DRS2/DNF subfamily of P-type ATPases.