Fig 1.
Synthetic pathways of major phospholipids in S. cerevisiae.
Magenta and blue arrows represent reactions of the CDP-DAG pathway and the Kennedy pathway, respectively. Black arrows represent interorganellar phospholipid transport.
Table 1.
Strains used in this study.
Table 2.
Plasmids used in this study.
Fig 2.
Overexpression of SFH1 restores the growth of the psd1Δ strain on lactate.
Cells were cultured in SD medium to logarithmic phase and were spotted on SD or SLac media in ten-fold serial dilutions and were incubated on SD medium for 2 days or on SLac medium for 7 days. (A) Growth of the psd1Δ strains overexpressing genes obtained from the screening. (B) Growth of psd1Δ strains overexpressing genes encoding Sec14 family proteins, Sec14 and Sfh1–Sfh5. (C) Growth of the psd1Δ strains overexpressing SFH1 mutants, sfh1S175I,T177I, sfh1R61A,T238D, sfh1L179W,I196W, and sfh1L179W,I196W. (D) Growth of psd1Δect1Δ overexpressing SFH1. (E) Growth of psd1Δpsd2Δ overexpressing SFH1. Etn was added to final concentration of 1 mM.
Fig 3.
Overexpression of SFH1 restores the phospholipid compositions and MRC IV protein level of the psd1Δ strain.
(A) Mitochondrial phospholipid composition of psd1Δ overexpressing SFH1. The psd1Δ strains harboring YCp111-PSD1, YEplac181, or YEp181-SFH1 were cultured in semi-synthetic lactate medium to late logarithmic phase, and mitochondria were purified by sucrose density gradient. Lipids were extracted by the Bligh and Dyer’s method, separated by thin-layer chromatography, and analyzed as described in the Material and Methods. Data are the means of three independent assays. Error bars represent S.E. **, p < 0.005 (two-tailed Student’s t-test) (B) Cellular phospholipid composition of psd1Δ overexpressing SFH1. The psd1Δ strains harboring YCp111-PSD1, YEplac181, YEp181-SFH1, or YEp181-SFH1S175I,T177I were cultured in semi-synthetic lactate medium to late logarithmic phase. Lipids were extracted and analyzed as described above. Data are the means of three independent assays. Error bars represent S.E. **, p < 0.005 (two-tailed Student’s t-test).
Fig 4.
SFH1 does not activate or increase Pss1 or Psd2 of the psd1Δ strain.
(A) Addition of recombinant Sfh1 did not potentiate Pss1 activity in vitro. Purified ER fraction from psd1Δpsd2Δ (closed symbols) or pss1Δ (open symbols) was mixed with [3H]L-serine in the presence (circles) or absence (triangles) of His8-Sfh1, and incubated at 30°C for 15, 30, 45, and 60 min. Pss1 activity was measured as described in Materials and methods. Data are the means of three independent assays. Error bars represent S.E. Note that the symbol of the Pss1 activity of the pss1Δ in the absence of His8-Sfh1 is close to that of the pss1Δ in the presence of His8-Sfh1. (B) Overexpression of SFH1 did not potentiate Psd2 activity. Postnuclear supernatant (PNS) (10 μg protein) prepared from psd1Δ or psd1Δpsd2Δ harboring SFH1 overexpression plasmid or empty vector was mixed with reaction solution containing 16:0–6:0 NBD-PS (10 μM) and incubated at 36°C for 15 min. Lipids were extracted by the Bligh and Dyer’s method and separated by TLC. NBD-PS was quantitated as described in Materials and methods. Data are the means of three independent assays. Error bars represent S.E. n.s., not significant. (C) and (D) Overexpression of SFH1 did not increase the protein levels of Pss1 and Psd2. Pss1-EGFP (C) and Psd2-FLAG (D) were expressed by low-copy vectors in psd1Δ harboring SFH1 overexpression plasmid or an empty vector. Protein levels were evaluated by immunoblot utilizing anti-GFP, anti-FLAG, and anti-Pgk1 antibodies. Image analysis of immunoblots signal intensities were carried out using Image J. Data are the means of three independent assays. Error bars represent S.E. n.s., not significant.
Fig 5.
Sfh1 binds phospholipids in vivo and transfers PS between liposomes in vitro.
(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.
Fig 6.
Sfh1 localizes in the cytosol, nucleus, and endosome, Golgi, and/or vacuole.
(A) Sfh1-EGFP localizes in the cytosol and nucleus. Fluorescence microscopic observation of W303-1A cells overexpressing SFH1-EGFP cultured in SD medium to logarithmic phase. (B) Sfh1-HA localizes in the cytosol and endosome, Golgi, and/or vacuole. The cell extract of psd1Δ overexpressing SFH1-HA cultured in SD medium was fractionated by sucrose density gradient centrifugation. Distributions of Sfh1-HA and organelle marker proteins were evaluated by immunoblot.
Fig 7.
Deletion of SFH1 impairs the growth of the psd1Δ strain on lactate.
(A) Growth of psd1Δ expressing SFH1. Cells were cultured in SD medium to logarithmic phase and were spotted on SD or SLac medium in five-fold serial dilutions and were incubated on SD medium for 2 days or on SLac medium for 10 days. (B) Growth of psd1Δsfh1Δ on lactate. Cells cultured in SD medium to logarithmic phase were harvested and suspended in distilled water. Cells of 1 x 10−5 OD600 unit were spread on SLac medium and incubated for 7 days. Colony diameter was measured and analyzed using Image J. *** represents p < 5e-8 by Man-Whitney U test. (C) Cellular phospholipid composition of psd1Δ and psd1Δsfh1Δ cultured in SD medium. Lipids were extracted by the Bligh and Dyer’s method and were analyzed as described in the Material and Methods. Data are the means of three independent assays. Error bars represent S.E. p values of two-tailed Student’s t-test are shown.
Fig 8.
ERMES complex is important for recovery of growth of psd1Δ on lactate by SFH1.
(A) psd1Δmdm34Δ containing YCp33-MDM34 with YCp11-MDM34, YCp111-PSD1, YEp181-SFH1, or YEplac181 was cultured in SD medium to logarithmic phase and were spotted on SD or SLac medium with or without 0.5 mM 5-FOA in five-fold serial dilutions. Strains were incubated on SD or SLac medium without 5-FOA for 2 or 7 days, and on SD or SLac medium with 5-FOA for 7 or 10 days, respectively. Slight growth of psd1Δmdm34Δ containing YCp11-MDM34 and YEplac181 on SLac medium in the presence of 5-FOA was probably due to longer incubation than that on SLac medium in the absence of 5-FOA. (B)–(D) Cells were cultured in SD medium to logarithmic phase and 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.
Fig 9.
A model of phospholipid transport by Sfh1.
Sfh1 may enhance transfer of PS synthesized in the ER to the endosome, Golgi, and vacuole, where PS is decarboxylated to PE by Psd2. Sfh1 may shuttle PE back to the ER, relieving the product inhibition of Psd2 by PE. PE is transferred from the ER to the mitochondria, possibly by ERMES complex.