<i>SFH1</i> does not activate or increase Pss1 or Psd2 of the <i>psd1</i>Δ 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.