Screening for novel regulatory factors of sphingolipid metabolism.

(A) de novo sphingolipid biosynthesis pathway in yeast, Saccharomyces cerevisiae. The pathway and proteins responsible for the synthesis of yeast sphingolipids are shown. IPC, inositol phosphorylceramide; MIPC, mannosylinositol phosphorylceramide; M(IP)2C, mannosyldiinositol phosphorylceramide. Myriocin (Myr) and aureobasidin A (AbA) inhibit the indicated steps of the sphingolipid biosynthesis pathway. (B) Schematic representation of screening for novel regulatory factors of sphingolipid metabolism by using the Myr-sensitive phenotype of lip1-1 cells. (C) Schematic representation of the TORC2-Ypk1-dependent regulatory mechanism of sphingolipid biosynthesis. (D) Wild-type and lip1-1 cells carrying an indicated plasmid were spotted at a 10-fold serial dilution on YPD supplemented with (0.2 µM) or without Myr, in the presence (20 µg/mL) or absence of doxycycline (Dox). (E) The diploid homozygous knockout cells of MLM genes (ypk1Δ, lsp1Δ, com2Δ, tif3Δ, and stm1Δ) were spotted at a 10-fold serial dilution on YPD supplemented with 1.0 µM Myr. (F, G) Wild-type (COM2-13Myc) and Ptetoff-lip1-1 cells were grown to mid-log in SD (+All) liquid medium in the presence (+) or absence (-) of Dox (20 µg/mL) and treated under the respective conditions for 3 hrs. Cells were then harvested and total cell lysates were prepared. The lysates were resolved by SDS-PAGE and immunoblotted with anti-Myc or anti-G6PDH antibodies, to detect Com2-13Myc or G6PDH (loading control), respectively. Western blot experiments were independently replicated three times. Values for Com2 expression level are expressed as mean ± SD (n≥3).

Multi-copy suppressor for lip1-1-myriocin sensitivity (MLM genes)

Com2 functions upstream of the TORC2-Ypk1 pathway.

(A) Schematic representation of Com2, with the positions of the predicted AGC-kinase-dependent phosphorylation sites and two Zinc-finger domains highlighted. (B) Characterization of ypk1-as ypk2Δ mutant. Wild-type, the ATP-analogue sensitive allele ypk1L424A, ypk1-as ypk2Δ cells and the wild-type allele of Ypk1, YPK1WT ypk2Δ cells were spotted on YPD containing DMSO (Mock) or indicated concentration of 3MB-PP1 and incubated for 3 days at 26 °C. ypk1-as ypk2Δ cells exhibit growth defect by limiting of Ypk1-activity by addition of 3MB-PP1. (C) Wild-type cells, YPK1WT ypk2Δ cells and ypk1-as ypk2Δ cells are spotted on YPD and YPD containing 0.7 μM myriocin and incubated for 2 days at 26 °C. ypk1-as ypk2Δ cells exhibit Myr-sensitive without inhibition by addition of 3MB-PP1. (D) Wild-type YPK1WT ypk2Δ cells and analogue-sensitive mutant YPK1L424Aypk2Δ cells carrying the plasmid expressing Com2-13Myc were treated with 3MB-PP1 for the indicated time-periods and then treated with 1.0 µM Myr for 2 hrs. The lysates were then resolved using phos-tag or normal SDS-PAGE and immunoblotted with anti-Myc, anti-FLAG, or anti-G6PDH antibodies, to detect Com2-13Myc, 3FLAG-Lag1, or G6PDH (loading control), respectively. (E) Wild-type (BY4743) and knockout homozygote diploid cells of ypk1Δ or com2Δ carrying an indicated multi-copy plasmid were spotted at a 10-fold serial dilution on YPD supplemented with the indicated concentrations of Myr.

Ypk1 is expressed in a Com2-dependent manner.

(A) Proposed model for the mechanism by which sphingolipid depletion increases Com2 expression, induces YPK1 transcription, and activates the TORC2-Ypk1 pathway to promote sphingolipid biosynthesis. (B) Wild-type and com2Δ cells were grown to mid-log phase in SD liquid medium and then treated with 1.0 µM Myr. The cells were then harvested, and total cell lysates were prepared from them. The lysates were resolved using SDS-PAGE and immunoblotted with anti-Myc, anti-Ypk1, anti-phospho-Ypk1T662, or anti-G6PDH antibodies, to detect Com2-13Myc, Ypk1, TORC2-dependent phosphorylation site (hydrophobic motif) of Ypk1 (p-Ypk1T662) or G6PDH (loading control), respectively. (C) The time-dependent increase in Com2 expression following Myr treatment was quantified in wild-type cells. Experiments were performed three times independently. Com2 levels were normalized to the internal control G6PDH, with the value at 0 min of Myr treatment set to 1. Data are presented as the mean ± SD from three independent experiments. Asterisks indicate statistically significant differences compared with the 0 min time point as determined by one-way ANOVA followed by Tukey’s multiple comparisons test (****p<0.0001). (D, E) To examine time-dependent changes in Ypk1 or phosphorylated Ypk1 at T662 (p-Ypk1T662) in wild-type and com2Δ cells, the levels of Ypk1 or p-Ypk1T662 at each time point after Myr treatment were normalized to the loading control G6PDH and then further normalized to the value at 0 min before Myr treatment in wild-type cells (set to 1). Data are presented as the mean ± SD from three independent experiments. Asterisks indicate statistically significant differences as determined by two-way ANOVA followed by Sidak’s multiple comparisons test (**p<0.01, ***p<0.001, ****p<0.0001). (F) To examine time-dependent changes in phosphorylated Ypk1 at T662 (p-Ypk1T662) in wild-type and com2Δ cells, the levels of p-Ypk1T662 at each time point after Myr treatment were normalized to total Ypk1 and then further normalized to the value at 0 min before Myr treatment in wild-type cells (set to 1). Data are presented as the mean ± SD from three independent experiments. Asterisks indicate statistically significant differences as determined by two-way ANOVA followed by Sidak’s multiple comparisons test (**p=0.0092). (G) Wild-type, slm1tsslm2Δ, tor2ts, and ypk1tsypk2Δ cells were grown to mid-log phase in SD liquid medium at 26°C and then shifted to 37°C for 30 min and treated with 1.0 μM Myr. The cells were then harvested at the indicated time-point and total cell lysates were prepared from them. The lysates were resolved using SDS-PAGE and immunoblotted with anti-Ypk1, anti-Myc, anti-G6PDH antibodies, or anti-phospho-Ypk1T662 antibodies, to detect Ypk1, Com2-13Myc, G6PDH (loading control), or TORC2-dependent phosphorylation of Ypk1 (p-Ypk1T662), respectively.

Overexpression of Com2 causes increased expression of Ypk1.

(A) The chromosomal COM2 promoter (PCOM2) was replaced by a Tet-off promoter and GFP was fused to the N-terminus of Com2, to generate the Ptet-off-GFP-COM2 strain. (B) Wild-type, com2Δ, and Ptet-off-GFP-COM2 cells were spotted at a 10-fold serial dilution on YPD supplemented with 0.6 or 1.0 µM Myr, in the presence (20 µg/mL) (+) or absence (-) of Dox. (C) YPK1 and LCB1 possess a putative Com2-binding site (CBS) in the promoter region. LacZ reporter plasmids containing the wild-type or Com2-binding site (CBS)-deleted promoter regions of YPK1 or LCB1 were used to assess Com2-dependent transcriptional regulation. (D) Ptet-off-GFP-COM2 cells were grown to mid-log phase in SD liquid medium in the presence (+) or absence (−) of doxycycline (Dox) and then treated with 1.0 µM Myr for 2 hrs. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-Com2, anti-Ypk1, anti-Myc, or anti-G6PDH antibodies to detect GFP-Com2, Ypk1, Lcb1-13Myc, or G6PDH (loading control), respectively. A representative immunoblot is shown. The full set of immunoblots from six independent experiments is provided in Figure 4—figure supplement 2. (E) Quantification of Ypk1 and Lcb1 protein levels shown in Figure 4—figure supplement 2. Band intensities were quantified using ImageJ. Data are presented as the mean ± SD from six independent experiments. Statistical significance was assessed using paired t-test (Ypk1, p=0.0071; Lcb1, p=0.0151). The right panel indicates paired differences with mean difference and 95% confidence intervals. (F) Ptet-off-GFP-COM2 cells carrying pPYPK1-LacZ, pPYPK1CBSΔ-LacZ or pPLCB1-LacZ, pPLCB1CBSΔ-LacZ plasmid were grown to mid-log phase in SD liquid medium, in the presence (+) or absence (-) of Dox. Lysates were obtained from the indicated samples and assayed for β-galactosidase activity. Data are presented as the mean ± SD from three independent experiments. ***p<0.001, as assessed using one-way ANOVA, with Tukey multiple comparison tests.

The Com2-binding site in the YPK1 promoter is required for both proper expression and TORC2-dependent phosphorylation of Ypk1.

(A) A schematic representation of the generation of the PYPK1-Com2 binding site–deleted (PYPK1-ΔCBS) strain using CRISPR-Cas9-mediated genome editing. The Cas9-gRNA complex introduces a double-strand break near the putative Com2 binding site (CBS) in the chromosomal YPK1 promoter (PYPK1). A donor DNA fragment lacking the CBS (PYPK1ΔCBS) is simultaneously introduced and integrated into the chromosome by homologous recombination, resulting in deletion of the CBS from the endogenous YPK1 promoter. (B) Wild-type, com2Δ, and PYPK1-CBSΔ cells were spotted at a 10-fold serial dilution on YPD supplemented with or without 0.6 µM Myr. (C) Wild-type, com2Δ, PYPK1ΔCBS, and Ptet-off-GFP-COM2 cells were grown to the exponential phase, diluted to OD600=0.1, and grown in YPD liquid medium treated with or without 0.6 µM Myr, in the absence (-) or presence (+) of Dox (20 µg/mL), in a microtiter plate. (D, E, F, G) The indicated cells were grown to mid-log phase in SD liquid medium and treated with 1.0 µM Myr at 26°C for 3 hrs. Cells were then harvested and whole cell lysates were prepared. Whole cell lysates were separated by SDS-PAGE and immunoblotted with anti-Myc, anti-Ypk1, anti-phospho-Ypk1T662 or anti-G6PDH antibodies to detect Com2-13Myc, Ypk1, p-Ypk1T662 or G6PDH (loading control), respectively. The amounts of Com2-13Myc, Ypk1, and p-Ypk1T662 in wild-type cells (containing Myr) were normalized by the amount of G6PDH. Data represents the mean ± SD of three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (*p<0.05, **p<0.01, ns: no significant difference). (H) TLC analysis of sphingolipids extracted from wild-type, com2Δ, PYPK1-CBSΔ, and Ptet-off-GFP-COM2 cells. Cells were grown to mid-log phase in YPD liquid medium, in the presence (+) or absence (-) of Dox (20 µg/mL) and treated with or without 0.2 µM Myr for 3 hrs. Complex sphingolipids were analyzed using TLC (left). The level of total complex sphingolipids in the wild-type cells was taken as 100% and each value is displayed as a graph (right). The data have been represented as mean ± SD of four independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (*p<0.05, ***p =0.0002, and ****p<0.0001).

Com2 expression is regulated by sequences within the ORF region.

(A) Schematic representation of Com2 expression plasmids carrying stepwise deletions in the 5′ upstream promoter region or truncations of the Com2 open reading frame (ORF) from either the N-terminal or C-terminal side. (B) The promoter- or ORF-deletion mutants of Com2 shown in (A) were transformed into com2Δ cells, and functional complementation of the Myr sensitivity of the com2Δ strain was assessed by spot assays using 10-fold serial dilutions on YPD medium or YPD medium supplemented with 0.6 µM Myr. (C) Expression of Com2-13Myc variants carrying stepwise deletions in the 5′ upstream promoter region of COM2 was examined in untreated cells or cells treated with 1.0 µM Myr for 3 hrs. (D) Expression of Com2-13Myc variants carrying N-terminal truncations of the ORF was analyzed under the same conditions as in (C). (E) Expression of Com2-13Myc variants carrying C-terminal truncations of the ORF was analyzed under the same conditions as in (C). For (C-E), com2Δ cells expressing 13Myc-tagged Com2 wild-type or deletion variants were grown to mid-log phase in SD (-Ura) liquid medium at 26°C, treated with 1.0 µM Myr for 3 hrs, and lysed. Proteins were separated by SDS-PAGE and immunoblotted with anti-Myc, anti-Ypk1, or anti-G6PDH antibodies to detect Com2-13Myc, Ypk1, or G6PDH (loading control), respectively.

Com2 is regulated by ubiquitin-proteasome system-mediated degradation in a manner dependent on intracellular sphingolipid levels.

(A) Sphingolipid biosynthetic pathway and perturbations at each step (Myr, PHS bypass, lip1-1 +Dox, aur1-19 +AbA, csg2Δ, ipt1Δ). (B) Immunoblot of Com2 and Ypk1 in wild-type cells treated with 1.0 µM Myr for 3 hrs, followed by 10 µM PHS for 3 hrs. G6PDH, loading control. (C-E) Quantification of (B): Com2 (C), Ypk1 (D), and p-Ypk1T662 (E). One-way ANOVA with Tukey’s test. (F) PHS-induced Com2 degradation in wild-type and sphingolipid-pathway-perturbed cells. Log-phase cultures were pretreated with 1 µM Myr for 3 hrs, then PHS for 3 hrs. lip1-1 was cultured with Dox (20 µg/mL) from the preculture; aur1-19 was pretreated with AbA (20 µg/mL) for 15 min before PHS. Immunoblots for Com2, Ypk1, and p-Ypk1T662; G6PDH, loading control. (G, H) Quantification of (F). One-way ANOVA with Tukey’s test. (I) Proteasome inhibition stabilizes ubiquitinated Com2. Wild-type cells depleted of sphingolipids by Myr (3 hrs) were incubated ±MG132 (50 µM) and bortezomib (100 µM) for 30 min, followed by PHS (10 µM). Samples were collected at 0-60 min and immunoblotted with anti-Myc (Com2-13Myc) and anti-G6PDH. (J) Quantification of (I) (normalized to G6PDH). Two-way ANOVA with Sidak’s test. (K) Model: Myr stabilizes Com2 to promote YPK1 expression and sphingolipid biosynthesis, whereas under +PHS conditions, Com2 is rapidly degraded via the ubiquitin-proteasome system. Quantified data (C-E, G-H, J) are mean ± SD (n=3). *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001; n.s., not significant.

Ubiquitination sites required for sphingolipid-dependent proteasomal degradation of Com2 and the role of Com2 phosphorylation.

(A) com2Δ cells expressing wild-type Com2 or Com2 (Δ2-40) were treated with 1.0 µM Myr for 3 hrs, then with PHS for 3 hrs; Com2 levels were analyzed by immunoblotting (G6PDH, loading control). (B) Schematic of predicted ubiquitination and phosphorylation sites in Com2. (C) com2Δ cells expressing wild-type Com2 or the indicated lysine mutants (K23,35R, K51R, K23,35,51R) were treated with 1.0 µM Myr for 3 hrs, then 10 µM PHS for 3 hrs; Com2 levels after Myr and after PHS were analyzed by immunoblotting (G6PDH, loading control). (D, E) Immunoblot (D) and quantification (E) comparing wild-type and K23,35,51R Com2 before and after PHS (n=3; one-way ANOVA with Tukey’s test; *p<0.1; n.s.). (F) Com2 phosphorylation was assessed ±λPPase in cells expressing wild-type Com2 or K23,35,51R after Myr→PHS treatment; immunoblotting for Com2 (G6PDH, loading control). (G) Phos-tag SDS-PAGE of Com2 phosphorylation-site mutants (ST-A3, ST-A5, ST-A7, ST-A8, ST-A10) based on mobility shifts. (H) Immunoblot analysis of Com2 levels in the indicated phosphorylation-site mutants before and after Myr (3 hrs) → PHS (3 hrs) treatment (G6PDH, loading control). (I, J) Immunoblot (I) and quantification (J) comparing wild-type and ST-A10 Com2 before and after PHS (n=3; one-way ANOVA with Tukey’s test; *p<0.1; n.s.).

Model for sphingolipid (SL) level-dependent regulation of Com2 degradation via the ubiquitin-proteasome system.

When intracellular SL levels are low, degradation of Com2 is suppressed, allowing Com2 to induce the expression of its target genes, such as YPK1 and LCB1, which harbor Com2-binding sites (CBSs) in their promoter regions, thereby promoting sphingolipid biosynthesis. In contrast, when intracellular SL levels are sufficient, complex sphingolipids such as MIPC, M(IP)2C are sensed by an as-yet-unidentified sphingolipid sensor, leading to phosphorylation of Com2, presumably by an unidentified kinase. This phosphorylation promotes ubiquitination of Com2 and its subsequent degradation by the proteasome.

Strains used in this study

Plasmids used in this study

Key resources table