Sfh1 binds phospholipids <i>in vivo</i> and transfers PS between liposomes <i>in vitro</i>.

(A) Phospholipids bound to Sfh1-ZZ and Sfh1S175I,T177I-ZZ. The wild-type strains harboring YEp181-SFH1-ZZ, YEp181-SFH1S175I,T177I-ZZ or YE181-SFH1 were cultured in SD medium to logarithmic phase. Proteins were purified from S100 fractions (4 mg protein) prepared from these cells. Co-purified lipids were extracted and analyzed by ESI-MS/MS. Data are the means of three independent assays. Error bars represent S.E. p values of two-tailed Student’s t-test are shown. (B) A schematic diagram of fluorescent dequenching assay. See Materials and Methods for details. (C) and (D) NBD-PS dequenching assay by Sfh1 and Sfh1S175I,T177I mutant were measured. Donor liposome (DOPC:DOPE:NBD-PS:Rhod-PE = 68:22:8:2 (mol%)) and acceptor liposome (DOPC:DOPE:soyPI:DOPS = 50:10:25:15 (mol%)) were mixed to final concentration of 25 μM and 250 μM, respectively. Fluorescence of NBD were chased at room temperature immediately after the addition of proteins or buffer. NBD fluorescence intensities were set to 0 at 0 s. Data are the means of three independent assays. Error bars represent S.E. (C) Proteins were added to final concentration of 800 nM. (D) NBD-PS dequenching assay by Sfh1 of various concentration (0, 400, 800, and 1200 nM). (E) Simultaneous SFH1 overexpression recovers the growth of psd1Δ overexpressing PSS1 on lactate. Cells were cultured in SD medium to logarithmic phase, and were spotted on SD or SLac medium in ten-fold serial dilutions and were incubated on SD medium for 2 days or on SLac medium for 7 days.