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), sec12ts (light gray), and svf1Δsec12ts (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.Svf1V12D-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.Svf1V12D-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.Svf1V12D-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.

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), sec12ts (light gray), and svf1Δsec12ts (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.Svf1V12D-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.Svf1V12D-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.Svf1V12D-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.

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