Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface

Abstract

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.

Figure 1.

SVF1 interacts genetically with yeast genes involved in ceramide transport. (a and b) A model for vesicular and non-vesicular ceramide transport in yeast under normal growth and ER stress conditions (b). (c) Histogram of correlation coefficients based on the genetic profiles of yeast mutants in chemical genetic screens. Data was extracted from Hillenmeyer et al. (2008). (d) Tetrad analysis of svf1Δosh3Δosh4Δ (green, yellow, and red, respectively) mutants crossed with nvj2Δosh2Δ (orange and blue, respectively). (e) Serial dilutions of WT, svf1Δ, nvj2Δ, and svf1Δnvj2Δ on YPD plates (control) and YPD plates containing 0.5 µM myriocin.

Figure S1.

Analysis of SVF1 genetic interactions with other potential ceramide transfer protein. (a) Quantification of tetrad analysis. Relative colony sizes of tetrads of diploid osh2Δosh3Δosh4Δsvf1Δ cells shown as fold change from WT tetrads. Error bars represent standard deviations. (b) Quantification of tetrad analysis. Relative colony sizes of tetrads of diploid osh2Δosh3Δosh4Δnvj2Δ shown as fold change from WT tetrads. Error bars represent standard deviations. (c) Tetrad analysis of svf1Δ (green) mutants crossed with sur2Δ (red). (d) Tetrad analysis of diploid svf1Δydr222wΔylr225cΔsur2Δ cells. svf1Δ (green); ydr222wΔ (blue); ylr225cΔ (yellow); sur2Δ (red). (e) Helical wheel representation of the first 18 amino acids of Svf1. The projection was generated by the Heliquest software (http://heliquest.ipmc.cnrs.fr). (f) Helical wheel representation of the first 18 amino acids of Ydr222w. (g) Helical wheel representation of the first 18 amino acids of Ylr225c.

Figure 2.

Svf1 dynamically colocalizes with the cis-Golgi. (a) Svf1-GFP was expressed in cells expressing either Mnn9-mKate (cis-Golgi), Sec7-mKate (trans-Golgi), Vps4-3xmCherry (endosome) or HDEL-dsRed (ER). Line scans (right) of the indicated regions of Svf1-GFP (green) and the respective co-expressed organelle markers (magenta). Scale bar = 5 µM. Scale bar inlays = 1 μM. (b) Co-localization of Svf1-GFP and 3xmCherry tagged Mnn9 imaged every 0.9 s over a total time of 9.9 s to confirm the localization to the cis-Golgi. Scale bar = 5 µM. (c) A model of the two interchanging states, cytosolic and membrane bound, of Svf1 in the cell.

Figure S2.

Tetrad dissections of N- and C-terminal tagged SVF1. (a) Tetrad analysis of a marker less generated GFP-Svf1 strain crossed with sur2Δ. Tetrads are numbered 1–5. Spores of the tetrads are labeled A to D. (b) Tetrad analysis of a Svf1-GFP strain crossed with sur2Δ. Tetrads are numbered 1–5. Spores of the tetrads are labeled A to D.

Figure 3.

Svf1 possesses an N-terminal amphipathic helix important for the targeting and function of the protein. (a and b) Helical wheel representation of the first 18 amino acids with the hydrophobic amino acids shown in yellow and the hydrophobic moment shown by the arrow and expressed in µH above the arrow for the WT sequence and (b) the V12D mutant with the exchange of the hydrophobic valine to the charged aspartate (red). (c) Graphical representation of the full-length protein (WT, left; V12D mutant, right). Shown are the AH (yellow, 1–18 aa), the rest of Svf1 (orange, 19–481 aa) and the C-terminal GFP tag (green). (d) Co-localization of GFP tagged Svf1 expressed from a plasmid under control of the endogenous promoter with mCherry tagged Mnn9 for the WT (upper panel) and the V12D mutant (lower panel). Scale bar = 5 µM. Scale bar inlays = 1 µM. (e) Samples from the membrane fractionation according to 50 µg protein concentration were analyzed by Western blot. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the Svf1 localization either bound to membranes (P) or cytosolic (S) detected by an α-GFP antibody. The antibodies α-Pgk1 and α-Sec61 were used as loading controls for the cytosol and membrane fractions, respectively. (f) Tetrad analysis of the svf1Δpl.Svf1-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (g) Tetrad analysis of the svf1Δpl.Svf1_V12D-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (h) A model of Svf1 with the folded (bound Svf1) and unfolded (cyt. Svf1) N-terminal AH. (i) Tetrad analysis of the svf1∆pl.Svf1_L2E-GFP (blue and green, respectively) mutants crossed with sur2∆ (red). (j) Western blot analysis of Svf1-GFP in WT and mak3Δ mutant as described in e. (k and l) Helical wheel representation of the WT AH (k) and the G7A/G8A mutant AH (l) described as in Fig. 3 a shows no impact by the exchanges of glycines to alanines at the positions 7 and 8 on the hydrophobic moment of the AHs. (m) Co-localization of GFP tagged Svf1 expressed from a plasmid under the control of the endogenous promoter of Svf1 in a svf1Δ background (WT, upper panel; G7A/G8A mutant, lower panel), with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (n) Western blot analysis as described in e for Svf1_WT-GFP and Svf1_G7A/G8A-GFP. Source data are available for this figure: SourceData F3.

Figure S3.

Expression levels of plasmid expressed Svf1 are slightly altered. (a) Expression levels of Svf1-GFP constructs used in this study. Equal amounts of cells were lysed and analyzed by Western blotting using antibodies against the GFP-tag or Pgk1 as a loading control. NC = negative control/ WT strain; end. WT = Svf1 GFP tagged endogenously with GFP; pl. WT = Svf1-GFP expressed under its endogenous promoter from an integrative plasmid; V12D = Svf1_V12D-GFP expressed under its endogenous promoter from an integrative plasmid; G7A/G8A = Svf1_G7A/G8A-GFP expressed under its endogenous promoter from an integrative plasmid; L2E = Svf1_L2E-GFP expressed under its endogenous promoter from an integrative plasmid. (b) Helical wheel representation of the first 18 amino acids with the hydrophobic amino acids shown in yellow and the hydrophobic moment shown by the arrow and expressed in µH above the arrow for the L2E mutant with the exchange of the hydrophobic leucine to the charged glutamate (red). Source data are available for this figure: SourceData FS3.

Figure S4.

Analysis of Svf1 acetylation and analyses of Svf1 mutants. (a) Mass spectrometric analysis of the N-terminus of Svf1 showing that it is N-terminally acetylated. MS/MS spectrum extracted from MaxQuant data from a GFP pulldown of Svf1. Detected y-ions are labeled in red, b-ions are labeled in blue. The sequence showing all identified y- and b-ions is shown in the top right position. (b) Tetrad analysis of the svf1Δpl.Grh1_1-18aaSvf1_19-481-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (c) Evaluation of the binding of the AHs from WT, V12D mutant, and G7A/G8A mutant to the ER expressed as cytosol/ER ratio in %. Intensities of the GFP signal measured in cytosolic areas were divided by the intensities of the GFP signal at the ER measured in 0.065 µm². The signal of Sec63-Halo was used to differentiate between Cytosol and ER areas. n ≤ 48.

Figure 4.

The N-terminal amphipathic helix of Svf1 targets to the ER. (a) Graphical representation of the AH of Svf1 (yellow; WT, left; V12D mutant, middle; G7A/G8A mutant, right) tagged with GFP. (b) Co-localization of the GFP tagged AH expressed from a plasmid under the control of the promoter of Svf1 (WT, upper panel; V12D, lower panel) with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (c) Western blot analysis of the WT and the V12D AH (WT and AH_V12D, respectively) fused to GFP. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the localization of the GFP tagged AH (WT) and AH_V12D either bound to membranes (P) or cytosolic (S) detected by an α-GFP antibody. The antibodies α-Pgk1 and α-Sec61 were used as loading controls for the cytosol and membrane fractions, respectively. (d) Co-localization of the GFP tagged AH expressed from a plasmid under the control of the endogenous promoter of Svf1 (WT, upper panel; G7A/G8A, lower panel) with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (e) Western blot analysis of the WT and the G7A/G8A AH (AHWT and AHG7A/G8A, respectively) fused to GFP. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the localization of the GFP tagged AHWT and AHG7A/G8A either bound to membranes (P) or cytosolic (S) detected by an α-GFP antibody. The antibodies α-Pgk1 and α-Sec61 were used as loading controls for the cytosol and membrane fractions, respectively. (f and g) Helical wheel representation of the 1–18 amino acids of Grh1 as described for Svf1 in Fig. 3 a and (g) represented in the model in purple (right), which were fused to Svf1 (19–481 aa; orange) and a C-terminal GFP tag (green). (h) Co-localization of GFP tagged Svf1 expressed from a plasmid under the control of the endogenous promoter of Svf1 with mKate tagged Mnn9 (upper panel) and the fused protein with the AH of Grh1 (1–18 aa) and Svf1 (19–481 aa) expressed from a plasmid under control of the endogenous promoter of Svf1 with mKate tagged Mnn9 (lower panel). Scale bar = 5 µM. Source data are available for this figure: SourceData F4.

Figure 5.

Svf1 acts at the interface of the ER and the cis-Golgi. (a) Proteomic analysis of Svf1-GFP, expressed from a plasmid under the control of the endogenous promoter of Svf1 in a svf1Δ background, and mock treated WT cells is shown. Protein intensities are plotted against heavy/light SILAC ratios. Significant outliers are colored in red (P < 1⁻¹¹), orange (P < 1⁻⁴), or steel blue (P < 0.05), other proteins are shown in light blue. (b) Co-localization of GFP tagged Svf1 with mKate tagged Mnn9 (cis-Golgi) and Halo tagged Sec31 (ER, ER exit sites). (c) Evaluation of the co-localization of Svf1 dots (n = 100, triplicates) shown in d with either the cis-Golgi (Mnn9), Sec31 (ER, COPII vesicles), or both simultaneously. Scale bar = 5 µM. (d) Co-localization of GFP tagged Svf1 with Halo tagged Sec63 (ER marker) in a sec12^ts background under permissive (24°C, upper panel) and non-permissive (37°C, lower panel) temperature. Scale bar = 5 µM. (e) Co-localization of GFP tagged Svf1_G7A/G8A with Halo tagged Sec31 (ER marker) and mKate tagged Mnn9 in a sec23^ts background (upper panels) and in a sec17^ts background (lower panels) under permissive (24°C) and non-permissive (37°C) temperature. Scale bar = 5 µM. (f) Quantification of e (n = 60, triplicates).

Figure 6.

Lipidomic analysis shows an effect of Svf1 on sphingolipid metabolism. (a and b) Measurement of the different SP intermediates in WT (black), svf1Δ (gray), and sur2Δ (white) cells shows an alteration of (a) the LCB (LCB 18:0;2 and LCB 18:0;3) levels and (b) the ceramide (CER 44:0;3 and CER 44:0;4) levels represented in pmol/µg protein. (c) From the same measurement the levels of the complex SPs IPC (IPC 44:0;3 and IPC 44:0;4) and MIPC (MIPC 44:0;3 and MIPC 44:0;4) are shown as peak areas (arbitrary unit [a.u.]). (d–g) Measurements of the of the complex SPs IPC (IPC 44:0;3 [d] and IPC 44:0;4 [e]) and MIPC (MIPC 44:0;3 [f] and MIPC 44:0;4 [g]) in WT (black), svf1Δ (dark gray), sec12^ts (light gray), and svf1Δsec12^ts (white) cells at the restrictive temperature (37°C). Error bars represent standard deviation from mean. (h–k) Measurements of the of the complex SPs IPC (IPC 44:0;3 (h) and IPC 44:0;4 (i) and MIPC (MIPC 44:0;3 [j] and MIPC 44:0;4 [k]) in WT, svf1Δ, nvj2Δ, svf1Δnvj2Δ, osh2Δosh3Δosh4Δ, osh2Δosh3Δosh4Δ svf1Δ, osh2Δosh3Δosh4Δ nvj2Δ, and osh2Δosh3Δosh4Δ svf1Δ nvj2Δ cells. The color code is indicated below. Error bars represent standard deviation from mean. (l) Flux analysis shows the incorporation of ²H₆-inositol and ¹³C₃¹⁵N-serine into IPC species (44:0;3 and 44:0;4). Unlabeled IPC, inositol only labeled IPC (+²H₆), serine only labeled IPC (+¹³C₂¹⁵N) and double labeled IPC (+¹³C₂²H₆¹⁵N) are shown in svf1Δ, svf1Δpl.Svf1-GFP and svf1Δpl.Svf1_V12D-GFP at the time point t = −15 min and t = 90 min related to the addition of the tracers at t = 0 min. The IPC levels are expressed in pmol/0.4 OD units. (m) Flux analysis shows the incorporation of ¹³C₃¹⁵N-serine into PS species (34:2 and 34:1). Unlabeled PS and serine labeled PS (+¹³C₂¹⁵N) are shown in svf1Δ, svf1Δpl.Svf1-GFP and svf1Δpl.Svf1_V12D-GFP at the time point t = −15 min and t = 90 min related to the addition of the tracer at t = 0 min. The PS level is expressed in pmol/0.4 OD units. (n) Flux analysis shows the incorporation of ²H₆-inositol into PI 34:1 species. Unlabeled PI and inositol labeled PI (+²H₆) are shown in svf1Δ, svf1Δpl.Svf1-GFP and svf1Δpl.Svf1_V12D-GFP at the time point t = −15 min and t = 90 min related to the addition of the tracers at t = 0 min. The PI levels are expressed in pmol/0.4 OD units. (o) Serial dilutions of WT, svf1Δ, nvj2Δ, svf1Δnvj2Δ, dga1Δlro1Δare1Δare2Δ (Δ4), Δ4svf1Δ, Δ4nvj2Δ, and Δ4svf1Δnvj2Δ on YPD plates (control) and YPD plates containing 20nM Auroebasidin A (Aba). Bars represent mean ± SD from three independent samples.

Figure S5.

In vitro COP-II budding assays for lipidomic analysis. (a) Experimental setup of COPII budding assays. (b) Western blot analysis of COPII budding assays using antibodies against Sec22 (upper panels), Erv46 (middle panels), and GFP tagged Svf1 variants (lower panels). (c) Mass spectrometric analysis of ceramides from in vitro budded COP-II vesicles. The percentage of ceramide detected in the COPII vesicle fraction versus the sum of ceramides detected in both pelleted membranes and COPII vesicles is shown for WT Svf1 (black) and Svf1_V12D (gray) for experiments with COPII coat added and without COPII coat added (n = 3). (d) Western blot analysis of Gas1 in WT, svf1Δ, and emp24Δ cells. Only in emp24Δ the pre-form of Gas1 is detected. Source data are available for this figure: SourceData FS5.

Figure S6.

Model for sphingolipid metabolism and Svf1 co-localization with the IPC synthase. (a) Overview of the multi-pathway flux analysis. ¹³C₃¹⁵N-serine and ²H₆-inositol were added as tracers to exponentially growing cells. The potential incorporation into SP metabolites are shown in a simplified model of yeast SP metabolism. KDH, 3-ketodihydrosphingosine; DHS, dihydrosphingosine; PHS, phytosphingosine; PI, phosphatidylinositol; IPC, inositol phosphorylceramide; MIPC, mannosylinositol phosphorylceramide; M(IP)₂C, mannosyldiinositol phosphorylceramide as well as in phosphatidylinositol and phosphatidylserine. (b) Svf1-GFP (green) was expressed in cells expressing Mnn9-mKate (cis-Golgi, red) and Aur1-Halo (mid-Golgi, blue). Scale bar = 5 µM. (c) Qunatification of Svf1 dots co-localizing with Mnn9, Aur1, Mnn9, and Aur1, only Mnn9, and only Aur1. (n = 100 Svf1 dots, triplicates).

Figure 7.

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.

Figure S7.

Purification of Svf1 from yeast cells. (a) Svf1-FLAG overproduced from the GAL1 promotor was purified via FLAG tag and analyzed by SDS-PAGE. I, Input; FT, flow through; W1, wash 1; W2, wash 2; E1, eluate 1; E2, eluate 2; B, beads. (b) Svf1_V12D-FLAG overproduced from the GAL1 promotor was purified via FLAG tag and analyzed by SDS-PAGE. I, Input; FT, flow through; W1, wash 1; W2, wash 2; E1, eluate 1; E2, eluate 2; B, beads. (c) Svf1_G7AG8A -FLAG overproduced from the GAL1 promotor was purified via FLAG tag and analyzed by SDS-PAGE. I, Input; FT, flow through; W1, wash 1; W2, wash 2; E1, eluate 1; E2, eluate 2; B, beads. (d) Svf1_H273AH274A -FLAG overproduced from the GAL1 promotor was purified via FLAG tag and analyzed by SDS-PAGE. I, Input; FT, flow through; W1, wash 1; E1, eluate 1. (e) Mass photometry analysis of the purified proteins from a–d. Source data are available for this figure: SourceData FS7.

Figure 8.

Model for the proposed function of Svf1 in ceramide transport at the ER-Golgi interface. A model for vesicular and non-vesicular ceramide transport in yeast under normal growth conditions. Ceramides are transported by vesicular transport while Nvj2 resides in the NVJ. Under these conditions Svf1 transports ceramides in a non-vesicular pathway. Svf1 first picks up ceramide in the ER in a transient interaction with the organelle. Svf1 targets the cis-Golgi apparatus and releases ceramides that is than metabolized into cSPs.