Svf1 binds ceramide in a hydrophobic pocket between its two lipocalin domains. (a) AlphaFold prediction of the structure of Svf1. AlphaFold produces a per-residue confidence score (pLDDT) between 0 and 100 according to the color code. (b) Different visualization of the predicted structure of Svf1 with its two lipocalin domains colored in blue and in purple and the N-terminal AH color coded in orange. (c) Results of the molecular docking studies. A 44:0;4 ceramide (purple) can be accommodated in the hydrophobic cleft between the two lipocalin domains. Amino acids and the ceramide headgroup are shown as balls and sticks in the enlarged view. (d and e) Targeted lipidomic analysis of ceramide 44:0;4 and (e) PC 16:0/18:1 extracted from the purified proteins. Proteins were purified via a FLAG-tag and extracts were used for chloroform methanol extraction of co-purified lipids. Lipids co-purified with Svf1-FLAG (black), Svf1_V12D-FLAG (dark gray), Svf1_G7AG8A-FLAG (medium gray) and Svf1_H273AH274A-FLAG (light gray) are shown. Bars represent mean ± SD from four independent samples. (f) Cartoon model of the predicted structure of Svf1 with the AH helix colored in light orange and the small α-helical cap colored in dark orange. The two side chains of the histidines H273 and H274 are shown. (g) Co-localization of GFP tagged Svf1 expressed from a plasmid under control of the endogenous promoter with mKate tagged Mnn9 and Halo tagged Sec63 for the WT (upper panel) and the H273A H274A mutant (lower panel). Scale bar = 5 µM. (h) Tetrad analysis of the svf1Δpl.Svf1_H273AH274A-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). Source data are available for this figure: SourceData F7.