Fungi have two sphingolipid classes. A, predominant molecular species of GlcCer and IPC in P. pastoris with the structural features distinguishing the two sphingolipid classes highlighted. In addition, the enzymes introducing these structural features as well as the corresponding gene names or, where applicable, the names of the knock-out strains used in this study are given. B, commercially available LCBs used in the in vitro enzyme assays shown in Fig. 5 and supplemental Fig. S3. Lipid names are according to the recommendations of the International Lipid Classification and Nomenclature Committee (47). Sphinganine (commonly called dihydrosphingosine) is the initial LCB that serves as precursor for the biosynthesis of the other LCBs. Sphing-4-enine (sphingosine) is representative of the desaturated LCBs found in GlcCer, whereas 4-hydroxysphinganine (phytosphingosine) is the typical LCB of (G)IPCs.

Most animals, plants, and fungi have multiple Cer synthases that fall into distinct phylogenetic groups. S. cerevisiae has two Cer synthases, Lag1p and Lac1p, which share a high degree of sequence identity (73%). In contrast, most fungi have two Cer synthases with a much lower degree of sequence identity (≈30%) that fall into distinct phylogenetic groups. In the text, we refer to the enzymes orthologous to S. cerevisiae Lag1p/Lac1p as Lag1p (for longevity assurance gene; Ref. 50) and to the enzymes from the phylogenetic group that is missing in S. cerevisiae as Bar1p (for biocontrol agent resistance; Ref. 51). From the latter group, A. nidulans BarA and K. lactis Lac1p (the name is misleading because this enzyme in not an ortholog of S. cerevisiae Lac1p) were investigated previously (25, 51). It is evident that, with the exception of the six mammalian Cer synthases (52), there is no consensus regarding gene nomenclature. A systematic nomenclature for Cer synthases from all organisms is urgently needed. The phylogenetic tree was constructed using Tree Puzzle (53) from an alignment of full-length protein sequences generated with T-Coffee (54). Only genes that have been investigated experimentally are depicted. UniProt identifiers are as follows: 1, P38703; 2, P28496; 3, Q6CVA7; 4, C4QWW1; 5, Q5BAG6; 6, C4R2K3; 7, Q6CP21; 8, Q5B548; 9, Q6NQI8; 10, Q9LDF2; 11, Q9LJK3; 12, Q7Z139; 13, Q9GYR9; 14, Q96G23; 15, Q8IU89; 16, Q9HA82; 17, Q8N5B7; 18, Q6ZMG9; 19, Q9W423; 20, Q9XWE9; 21, P27544. A. thaliana, Arabidopsis thaliana; H. sapiens, Homo sapiens; C. elegans, Caenorhabditis elegans; D. melanogaster; Drosophila melanogaster.

Presence or absence of GlcCer in mutants of P. pastoris with altered activities of enzymes involved in sphingolipid biosynthesis. Lipids extracted from WT and mutant lines were separated by TLC in chloroform/methanol (85:15). Glycolipids were visualized by spraying with α-naphthol/sulfuric acid and subsequent heating to 160 °C. For interpretation of the results see the text. The WT and the knock-out mutants were grown in complete medium (YPD medium), whereas the GCS-overexpressing strains (right three lanes) were grown in minimal methanol medium to induce expression of the GCS. Overexpression of the GCS resulted in the appearance of an additional glycolipid band above the normal GlcCer band that turned out to be a mixture of steryl glucoside (SG) and atypical GlcCer species (see the text and Fig. 7). Biosynthesis of steryl glucoside is typical for P. pastoris cells grown in minimal medium. One representative of several experiments is shown.

Cer and GlcCer species can be classified into two groups that are distinguished by chain length of fatty acid and hydroxylation of LCB. Molecular species of Cer and GlcCer from WT and the knock-out strains bar1Δ, gcsΔ, and scs7Δ are grouped by the number of acyl carbons (assuming a C₁₈ LCB) as well as by the hydroxylation of the LCB (dihydroxy or trihydroxy) and the fatty acid (hydroxy or non-hydroxy). The quantities of Cer and GlcCer in the knock-out strains are given as mol % of the sum of all species relative to the WT so that a direct comparison between the strains is possible. Shown are averages and S.D. of three independent experiments.

Substrate preference of Bar1p matches the properties of the Cer pool with dihydroxy LCB and C₁₆/C₁₈ fatty acids. A, the substrate preference of Bar1p for different LCBs and acyl-CoAs was tested by a ceramide synthase assay with microsomes prepared from a lag1Δlac1Δ S. cerevisiae strain expressing FLAG-tagged P. pastoris Bar1p as its only Cer synthase (incubation time, 5 min). Non-hydroxylated acyl-CoAs with different chain lengths are compared on the left, whereas the preference for α-hydroxylated versus non-hydroxylated C₁₈ acyl-CoA is shown in the middle. Sphing-4-enine was used as the LCB. On the right, the preference for the LCBs sphinganine, sphing-4-enine, and 4-hydroxysphinganine is shown in combination with non-hydroxylated C₁₈ acyl-CoA. In the upper row, the substrate specificity was tested using both a single LCB and a single acyl-CoA per reaction (one reaction per combination), whereas in the lower row, substrate selectivity was tested by offering mixtures of the LCBs or acyl-CoAs to be compared against each other (one reaction per panel). Shown are averages and S.D. from three independent reactions. B, comparison of Cer species from two different S. cerevisiae strains: left, a control strain expressing c-Myc-tagged S. cerevisiae Lag1p; right, a strain expressing FLAG-tagged P. pastoris Bar1p as the only ceramide synthase. The Cer species are grouped by the number of acyl carbons, assuming a C₁₈ (upper row of carbon numbers) or a C₂₀ LCB (lower row; see supplemental Fig. S2), and by the hydroxylation of the LCB (dihydroxy or trihydroxy). Hydroxylation of the fatty acid is not taken into account because there were no significant differences between the strains. Shown are averages and S.D. of three independent experiments. The mol % of the sum of all Cer species was calculated separately for each strain so that the chain length distribution and the hydroxylation pattern, but not the absolute amounts, can be compared between the strains. 100% corresponds to 15 ± 2 nmol/g fresh weight for the Lag1p-expressing strain and 68 ± 8 nmol/g fresh weight for the Bar1p-expressing strain.

Δ4-Desaturation, but not Δ8-desaturation or C9 methylation, is essential for conversion of the Cer pool with dihydroxy LCB and C₁₆/C₁₈ fatty acids into GlcCer. Molecular species of Cer and GlcCer from WT and from the knock-out strains gcsΔ, c9Δ, delta8Δ, and delta4Δ are grouped by the desaturation of the LCB (d18:0, saturated; d18:1, monounsaturated; d18:2, diunsaturated; d18:1-9m, monounsaturated and C9-methylated; d18:2-9m, diunsaturated and C9-methylated) and by the hydroxylation of the fatty acid (hydroxy or non-hydroxy). Only Cer and GlcCer species with dihydroxy LCBs are shown because no species with a desaturated trihydroxy LCB could be detected. The acyl chain length of these species is almost exclusively 16 or 18 because nearly all Cer species with a C₂₄/C₂₆ fatty acid have a trihydroxy LCB (not included in this figure), and GlcCer species with a C₂₄/C₂₆ fatty acid occur only in trace amounts (Fig. 4). The quantities of Cer and GlcCer in the knock-out strains are given as mol % of the sum of all species relative to the WT so that a direct comparison between the strains is possible. Shown are averages and S.D. of three independent experiments.

Overexpression of GCS makes the Cer pool with trihydroxy LCB and C₂₄/C₂₆ fatty acids accessible for GlcCer biosynthesis. Molecular species of Cer and GlcCer from WT and from the overexpressing strains gcsΔGCS, delta4ΔGCS, and scs7ΔGCS are grouped by the number of acyl carbons (assuming a C₁₈ LCB) as well as by the hydroxylation of the LCB (dihydroxy or trihydroxy) and the fatty acid (hydroxy or non-hydroxy). The quantities of Cer and GlcCer in the overexpressing strains are given as mol % of the sum of all species relative to the WT so that a direct comparison between the strains is possible. Shown are averages and S.D. of three independent experiments.

Analysis of IPC, MIPC, and M(IP)₂C in P. pastoris strains lacking GlcCer. Molecular species of IPC, MIPC, and M(IP)₂C from WT and from the knock-out strains bar1Δ, gcsΔ, and scs7Δ are grouped by the hydroxylation of their fatty acids. Only species containing a trihydroxy LCB are shown. The WT strain contains in addition ≈11% of IPC species with a dihydroxy LCB in confirmation of a previous study (55). MIPC and M(IP)₂C species with a dihydroxy LCB were not detectable. The quantities of IPC, MIPC, and M(IP)₂C are given as mol % of the sum of all species relative to the WT so that a direct comparison between the strains is possible. Shown are averages and S.D. of three independent experiments. An absolute quantification was not possible because no standards were available.

Analysis of LCBs from WT P. pastoris. LCBs were released from sphingolipids by strong alkaline hydrolysis, converted into dinitrophenyl derivatives, and separated by HPLC. Upper panel, LCBs were released from Cer, GlcCer, and (G)IPCs of whole P. pastoris cells. The total LCBs consisted of ≈90% of the trihydroxy LCBs t18:0 and t20:0. Dihydroxy LCBs from the Bar1p-dependent Cer pool and from GlcCer made ≈10%. The left peak represents mainly t18:0 and a low proportion of d18:2, which cannot be separated by this method. Lower panel, LCBs, consisting of the three dihydroxy LCBs d18:1, d18:2, and d18:2-9m, were released from purified GlcCer from P. pastoris. Shown is one representative experiment of three.

Simplified model of sphingolipid biosynthesis in P. pastoris showing qualitative and quantitative routing of substrates into Cer, GlcCer, and IPC. Intermediates of the pathway are given with the predominant molecular species indicated underneath. The width of the arrows and the letters indicates the relative quantitative proportions of the end products of lipid biosynthesis in P. pastoris (not to scale). The compounds with a gray background were subjects of our analyses. The early steps of sphingolipid biosynthesis, the late steps of GIPC biosynthesis, and degradative and recycling pathways have been omitted. For simplicity, the model only shows species containing the dominating C₁₈ and C₂₄ fatty acids, but it should be considered that P. pastoris also synthesizes a smaller proportion of species containing C₁₆ and C₂₆ fatty acids.