Figure 6.

Cvm1 plays a role in sphingolipid homeostasis. (A) Diagram of the S. cerevisiae sphingolipid biosynthesis pathway, the enzymes involved, and the steps inhibited by the drugs myriocin and AbA. LCB, long-chain base. (B) Cells lacking Cvm1 are hypersensitive to myriocin. A WT strain, a strain carrying a CVM1 deletion, or three different clones carrying a CVM1 deletion transformed with a plasmid carrying CVM1 were spotted as seriated dilutions on YPD medium with or without 1 μM myriocin. (C) Cells with overexpressed CVM1 under the control of the TEF1 promoter are hyperresistant to myriocin. WT and CVM1 overexpression strains were spotted as seriated dilutions on YPD medium with or without 1.5 µM myriocin. (D) Cells lacking or overexpressing Cvm1 are hypersensitive to AbA. A WT strain, a CVM1 deletion strain, and a strain overexpressing CVM1 From the TEF1 promoter were spotted as seriated dilutions on YPD medium with or without 70 nM AbA. (E) MS-based lipidomics of whole-cell lysates of WT, Δcvm1, and CVM1 overexpression strains. Samples were normalized according to protein levels and an internal standard. For each lipid class, a ratio to the WT strain is shown. Five independent purifications were measured; bar graphs represent average ± SD, and the values for the independent samples are shown as diamonds. Samples that deviate significantly from the WT (P < 0.05) are marked with an asterisk. PA, phosphatidic acid; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; DG, diacylglycerol; TAG, triacylglycerol. (F and G) Targeted multiple-reaction monitoring MS lipid measurements of dihidrosphingosine (DHS), phytosphingosine (PHS), and ceramides (PHC) in whole-cell lysates of WT, Δcvm1 (F), and CVM1 (G) overexpression strains. Bars represent mean ± SD from four independent samples which are also shown as diamonds. Samples that deviate significantly from the WT (P < 0.05) are marked with an asterisk.