Introduction

Cell membranes are composed of lipid bilayers, which consist of three major types of membrane lipids: glycerophospholipids, sphingolipids, and sterols (mainly cholesterol in mammals and ergosterol in yeasts) (van Meer et al., 2008). The synthesis of lipid bilayers during membrane formation involves the coordinated assembly of multiple lipid species, which requires the coordination of different levels, including lipid synthesis, uptake, metabolism, and subcellular distribution (Breslow and Weissman, 2010; Nohturfft and Zhang, 2009). This homeostasis is important for the maintenance of cellular and tissue functions, and its dysfunction in humans can lead to numerous diseases, such as cancer or type-2 diabetes (Harayama and Riezman, 2018). The principal instruments for the global control of lipid metabolism are regulated by master transcriptional regulators, which target genes encoding metabolic enzymes and/or their regulators working in specific metabolic pathways (Nohturfft and Zhang, 2009).

For example, in yeast, the transcription factors Ino2/Ino4 function in the expression of several glycerophospholipid synthases (Kwiatek et al., 2020). Under logarithmic growth or inositol-depleted conditions, the repressor Opi1 is bound to the endoplasmic reticulum (ER) membrane in a phosphatidic acid (PA)-dependent manner, and Ino2/Ino4 promote transcription. However, in steady-state or inositol-rich conditions, the decrease in PA on the ER membrane causes Opi1 to translocate to the nucleus, thereby inhibiting Ino2/Ino4-dependent transcription and repressing glycerophospholipid synthesis (Ambroziak and Henry, 1994; Loewen, 2003; Loewen et al., 2004; White et al., 1991). In the cholesterol metabolism of animal cells, the membrane-bound transcription factors, SREBPs, are synthesized as inactive precursors that are anchored in the membrane of the ER under sterol-rich conditions (Hua et al., 1996). SREBPs are translocated to the Golgi apparatus in a COPII vesicle-dependent manner (Nohturfft et al., 2000, 1999, 1998), where it is sequentially cleaved by Site-1 and Site-2 proteases, detached from the membrane (Rawson et al., 1997; Sakai et al., 1998, 1996; Wang et al., 1994), and translocated to the nucleus, thus promoting the expression of genes involved in cholesterol metabolism, such as HMG CoA reductase, the rate-limiting enzyme in cholesterol metabolism (Goldstein et al., 2006; Nohturfft and Zhang, 2009). Thus, in glycerophospholipid and sterol metabolism, it is well known that the synthesis of these lipids is coordinately regulated by master transcriptional regulators, in both yeast and mammals. However, master transcriptional regulators of sphingolipid metabolism remain elusive (Breslow and Weissman, 2010).

Sphingolipids and their metabolites play key cellular roles as both structural components of membranes and as signaling molecules that mediate responses to physiological cues (Breslow and Weissman, 2010). Recent studies using budding yeast, Saccharomyces cerevisiae, as a model system have improved our understanding of metabolic enzymes and how these enzymes are regulated to ensure sphingolipid homeostasis (Breslow et al., 2010; Breslow and Weissman, 2010). The regulation of sphingolipid metabolism by the target of rapamycin (TOR) complex 2 (TORC2)-Ypk1 pathway has been studied in detail in yeast (Berchtold et al., 2012; Eltschinger and Loewith, 2016; Niles et al., 2012; Tabuchi et al., 2006). As a regulatory mechanism of sphingolipid biosynthesis, TORC2, in concert with Slm1/2, which was identified as a synthetic lethal gene along with MSS4 encoding a phosphatidylinositol 4-phosphate 5-kinase (Audhya et al., 2004; Tabuchi et al., 2006), phosphorylates Ypk1 upon depletion of complex sphingolipids at the plasma membrane (Berchtold et al., 2012; Niles et al., 2012). Phosphorylated Ypk1 further phosphorylates Orm1/2, which negatively regulates serine palmitoyl transferase (SPT) (Breslow et al., 2010; Gururaj et al., 2013; Roelants et al., 2011), which is the first step in sphingolipid metabolism. Thereafter, the phosphorylation of Orm1/2 through Ypk1 results in an increase in SPT activity. The TORC2-Ypk1 signal has also been reported to activate ceramide synthase by phosphorylating the N-terminal sites of the ceramide synthase catalytic subunits, Lag1/Lac1 (Muir et al., 2014). Together, TORC2-Ypk1-dependent signaling simultaneously upregulates the two most important steps in the sphingolipid biosynthetic pathway.

While investigating how reduced ceramide synthesis affects the TORC2-Ypk1 pathway using the ceramide synthase regulatory subunit mutant lip1-1, we found that lip1-1 is hypersensitive to myriocin (Myr), a specific inhibitor of SPT (Figure 1A) (Ishino et al., 2022).

Figure 1.

To identify novel regulators of sphingolipid metabolism, we performed a multicopy suppressor screen for genes that alleviate the Myr sensitivity of lip1-1 cells. This approach led to the identification of Com2, a C2H2-type zinc finger transcription factor that had not previously been linked to sphingolipid metabolism. Further analyses revealed that Myr-induced sphingolipid depletion resulted in increased Com2 expression, accompanied by elevated Ypk1 expression and enhanced TORC2-dependent phosphorylation of Ypk1. In com2Δ cells, both the Myr-induced upregulation of Ypk1 and its activation by TORC2 were markedly reduced. Moreover, deletion of the Com2-binding site (CBS) in the YPK1 promoter abolished these inductions, indicating that Ypk1 upregulation in response to sphingolipid depletion depends on Com2 and the CBS element in the YPK1 promoter. We further found that Com2 is rapidly degraded via the ubiquitin-proteasome system (UPS) upon addition of exogenous sphingolipid precursor, demonstrating that Com2 abundance is primarily controlled through UPS-dependent proteolysis in a sphingolipid-dependent manner.

Collectively, these findings suggest that Com2 acts as a master transcription factor that senses intracellular sphingolipid levels, regulates CBS-dependent target genes such as YPK1, and is itself controlled by sphingolipid-dependent degradation via the ubiquitin–proteasome system.

Results

A screen for novel regulatory factors involved in sphingolipid metabolism

We previously generated and characterized a P_tet-off-lip1-1 (lip1-1) strain in which ceramide synthesis decreased in the presence of doxycycline (Dox) (Ishino et al., 2022). Dox-treated lip1-1 cells are hypersensitive to myriocin (Myr), an inhibitor of serine palmitoyltransferase (SPT), the first step in sphingolipid biosynthesis (Figure 1A), presumably due to the synthetic effect of the Dox-dependent decrease in ceramide synthesis and inhibition of SPT. We hypothesized that overexpressing a protein involved in sphingolipid biosynthesis would confer Myr resistance to lip1-1 cells. To identify novel regulatory factors, we transformed lip1-1 cells with a multi-copy genomic library and selected transformants that grew on Myr-containing media (Figure 1B). Sequencing 27 Myr-resistant plasmids revealed 12 gene groups (MLM genes: Multi-copy suppressor for Lip1-1 Myr-sensitive phenotype, Figure1—figure supplement 1, Table 1). Among these, the YPK1 (MLM5), which encodes for a protein kinase downstream of the TORC2 signaling pathway, which is known to be involved in the regulation of sphingolipid metabolism (Figure 1C), was identified, thus validating that genes involved in the regulation of sphingolipid metabolism can be obtained employing this screening. Besides the LIP1 (MLM1), the COM2 (MLM2), which encodes a C2H2-type zinc finger transcription factor, was the most abundant (Figure 1D, Figure1—figure supplement 1, Table 1).

Table 1.

To clarify whether the obtained MLM genes are involved in the regulation of sphingolipid metabolism, we examined the Myr sensitivity of each gene-knockout homozygous diploid strain. Upon doing so, we found that com2Δ, as well as ypk1Δ, exhibited Myr sensitivity, while the other MLM gene-deficient strains did not (Figure 1E, Figure 1—figure supplement 2).

We further examined the phenotype of com2Δ in response to various drugs and stress conditions in addition to Myr. Our results showed that com2Δ exhibited higher sensitivity to Myr compared to the wild-type. However, it did not display sensitivity to aureobasidin A (AbA), an inhibitor of inositol phosphoceramide (IPC) synthesis, ergosterol synthesis inhibitors such as miconazole (MCZ) and terbinafine, or any other drugs and stress conditions tested (Figure1—figure supplement 3A-C). Unlike ypk1Δ, com2Δ does not exhibit sensitivity to AbA. This observation suggests that Com2 plays a more specialized role in regulating sphingolipid biosynthesis at an earlier, upstream step.

Next, we analyzed the expression levels of Com2 in wild-type cells under various drug and stress treatment conditions. We observed a remarkable upregulation of Com2 expression under conditions that reduced complex sphingolipid synthesis, such as in Myr-treated cells, AbA-treated cells or Dox-treated lip1-1 cells (Figure 1F, G). In contrast, inhibition of sterol synthesis did not lead to a significant increase in Com2 expression (Figure 1—figure supplement 3D, E). These findings indicate that Com2 expression is specifically upregulated in response to a reduction in complex sphingolipid levels.

Com2 functions upstream of TORC2-Ypk1 pathway

Overexpression of Com2 or Ypk1 suppressed the Myr sensitivity of lip1-1 (Figure 1D) and deletion of any of these genes conferred Myr sensitivity (Figure 1E), suggesting that these two proteins function in the same pathway involved in the regulation of sphingolipid metabolism. Com2 is a C2H2-type zinc finger transcription factor consisting of 443 amino acid residues with two zinc fingers at its C-terminus. In addition to that, several putative phosphorylation sites for AGC kinases, including Ypk1, were found in its amino acid sequence (Figure 2A) (Muir et al., 2014). Based on these observations, we hypothesized that Com2 is involved in sphingolipid metabolism downstream of Ypk1 and that Ypk1 impacts the phosphorylation and function of Com2.

Figure 2.

To test this hypothesis, we investigated the effect of limiting Ypk1 activity on the phosphorylation state of Com2. To this end, we employed ypk1-as ypk2Δ cells, which express an ATP analog-sensitive (AS) allele, Ypk1(L424A), and titrated down its activity by addition of an efficacious inhibitor, 1-(tert-butyl)-3-(3-methylbenzyl)-1H-pyrazole[3,4-d]pyrimidin-4-amoine (3-MB-PP1), which has no effect on the wild-type cells (Figure 2B) (Muir et al., 2014). As previously reported (Ishino et al., 2022; Muir et al., 2014), Myr treatment stimulated the phosphorylation of the 3FLAG-tagged Lag1 in wild-type cells, as verified using Phos-tag SDS-PAGE (Figure 2C, lanes 1-5 in the 3FLAG-Lag1 blot), but this effect was abolished in ypk1-as ypk2Δ cells (Figure 2C, lanes 7-11 in the 3FLAG-Lag1 blot), confirming the inhibition of Ypk1 activity. It is noted that the ypk1-as ypk2Δ mutant did not show Myr-dependent phosphorylation of Lag1 even in the absence of 3-MB-PP1 (Figure 2C, lane 11 in 3FLAG-Lag1 blot) and was also sensitive to Myr (Figure 2D), suggesting that this mutant appears to be a hypomorphic allele and treatment of 3-MB-PP1 completely blocks Ypk1 activity. In contrast to phosphorylation of Lag1, a 13Myc epitope-tagged Com2 was highly phosphorylated in both the wild-type and ypk1-as ypk2Δ cells (Figure 2C, lanes 1-5 and lanes 7-11 in the Com2-13Myc blot of Phos-tag gel), indicating that the phosphorylation state of Com2 is not dependent on Ypk1 activity. Notably, as shown in Figure 1F, G, Myr treatment increased Com2 expression in wild-type cells, and this effect was more pronounced in ypk1-as ypk2Δ cells treated with 3-MB-PP1.

Next, we performed an epistatic analysis between COM2 and YPK1, confirming that overexpressing each gene suppressed Myr sensitivity in the corresponding deletion strain. Overexpression of COM2 did not suppress the Myr sensitivity of ypk1Δ cells, whereas overexpression of YPK1 suppressed the Myr sensitivity of com2Δ cells (Figure 2E), indicating that Com2 functions upstream of Ypk1.

Taken together, these results suggested that Com2 functions upstream of the TORC2-Ypk1 pathway rather than downstream of it.

Com2 expression increases in response to a decrease in sphingolipids

Epistasis analysis suggested that COM2 functions upstream of YPK1 (Figure 2E). We noticed a putative Com2-binding site (CBS; 5′-CCCTAT-3′) exists in the YPK1 promoter (Siggers et al., 2014). Based on these observations, we hypothesized that Com2 regulates YPK1 expression in a sphingolipid-dependent manner (Figure 3A).

Figure 3.

To test this hypothesis, we performed a time-course analysis of Com2 and Ypk1 during Myr treatment in wild-type and com2Δ cells and assessed protein levels by immunoblotting. In wild-type cells, Myr-treatment caused a time-dependent increase in Com2, accompanied by an increase in total Ypk1 as well as enhanced TORC2-dependent phosphorylation of the Ypk1 hydrophobic motif (p-Ypk1^T662) (Figure 3B, lanes 1-5, Figure 3C-E). In contrast, in com2Δ cells, both the upregulation of Ypk1 and the Myr-dependent increase in p-Ypk1^T662 were strongly attenuated (Figure 3B, lanes 6–10, Figure 3D, E), indicating that these responses depend on Myr-induced Com2 expression. Notably, TORC2 activity was not reduced in com2Δ cells (if anything, it appeared elevated) (Figure 3F), suggesting that the diminished p-Ypk1^T662 signal primarily reflects decreased Ypk1 abundance rather than defective TORC2 activation.

TORC2 senses plasma membrane stress caused by a reduction in complex sphingolipids through the eisosome-associated adaptor proteins, Slm1/2, thereby activating Ypk1 to stimulate sphingolipid synthesis (Figure 1C) (Berchtold et al., 2012). Next, we investigated the contribution of the Slm-TORC2-Ypk1 pathway to the Myr-induced expression of Com2. We observed Myr-induced expression of Com2 even after blocking of the Slm-TORC2-Ypk1 pathway at non-permissive temperatures using each temperature-sensitive mutant (Figure 3G), indicating that the induction of Com2 expression upon decreased complex sphingolipids is due to a sensing pathway that is independent from the Slm-TORC2-Ypk1 pathway.

Taken together, we demonstrated that Com2 is upregulated in response to a decrease in sphingolipid synthesis, accompanied by upregulation of Ypk1 expression and its TORC2-dependent phosphorylation (Figure 3A).

Overexpression of Com2 causes increased expression of Ypk1 via a putative CBS in the YPK1 promoter

To further substantiate that Com2 acts upstream of Ypk1, we replaced the endogenous COM2 promoter with a tetracycline-repressible tet-off promoter by homologous recombination, generating a P_tet-off-GFP-COM2 strain in which Com2 expression can be controlled by Dox (Figure 4A). Using this strain, we assessed Myr sensitivity under conditions of GFP-Com2 overexpression or repression. Cells overexpressing GFP-Com2 (-Dox) were more resistant to Myr than wild-type cells (Figure 4B, upper panel), whereas repression of GFP-Com2 (+Dox) rendered cells as Myr-sensitive as com2Δ cells (Figure 4B, lower panel).

Figure 4.

A putative Com2-binding site (CBS) was identified in the YPK1 promoter (Figure 4C). In addition, a genome-wide search of the S. cerevisiae genome using the CBS consensus motif revealed a matching sequence in the promoter of LCB1, which encodes a catalytic subunit of SPT (Figure 4C; Figure 4—figure supplement 1). To test whether Com2 directly regulates YPK1 and LCB1 expression, we examined the effects of GFP-Com2 overexpression on their expression levels. Induction of GFP-Com2 overexpression (-Dox) markedly increased Ypk1 abundance, and also led to a significant, albeit more modest, increase in Lcb1 abundance (Figure 4D, E, Figure 4—figure supplement 2).

We next constructed lacZ reporters driven by the YPK1 or LCB1 promoter (Figure 4C) and assessed promoter activity by measuring β-galactosidase activity. Compared with conditions in which GFP-Com2 was repressed (+Dox), GFP-Com2 induction (-Dox) increased β-galactosidase activity ∼6-fold for the YPK1 promoter and ∼2.5-fold for the LCB1 promoter (Figure 4F). Notably, this Com2-dependent activation was abolished by deleting the CBS from each promoter, indicating that Com2 directly stimulates YPK1 and LCB1 transcription via the CBS motif. Collectively, these findings support a model in which Com2 contributes to sphingolipid metabolism by transcriptionally upregulating YPK1 and LCB1.

Com2 promotes sphingolipid synthesis, partly through the expression of Ypk1

To test whether Com2 directly regulates YPK1 transcription and how this regulation impacts sphingolipid metabolism, we used CRISPR-Cas9 genome editing to delete the putative Com2-binding site (CBS) from the endogenous YPK1 promoter, generating P_YPK1-ΔCBS cells (Figure 5A).

Figure 5.

We first assessed the Myr sensitivity of P_YPK1-ΔCBS cells. P_YPK1-ΔCBS cells were more sensitive to Myr than wild-type cells, but less sensitive than com2Δ cells or doxycycline-treated P_tet-off-GFP-COM2 cells (+Dox) (Figure 5B, C).

We next examined Ypk1 expression in P_YPK1-ΔCBS cells by western blotting. Under Myr-treated conditions, Com2-13Myc levels were comparable between wild-type and P_YPK1-ΔCBS cells, whereas Ypk1 levels were reduced in P_YPK1-ΔCBS cells relative to wild-type and were similar to those in com2Δ cells (Figure 5D–F). Consistently, TORC2-dependent phosphorylation at the hydrophobic motif (p-Ypk1^T662) was decreased in P_YPK1-ΔCBS cells compared with wild-type and was comparable to that in com2Δ cells (Figure 5D, G).

We then performed TLC analysis to examine how Com2-dependent regulation influences sphingolipid synthesis. Consistent with the Myr resistant phenotype, overexpression of GFP-Com2 (-Dox) significantly increased sphingolipid levels under both untreated (Figure 5H, lane 1 vs. lane 5) and Myr-treated conditions (Figure 5H, lane 6 vs. lane 10). In contrast, under Myr treatment, sphingolipid levels were substantially reduced in com2Δ cells and in GFP-Com2-repressed (+Dox) cells compared with wild-type (Figure 5H, lane 6 vs. lane 7 or 9). In Myr-treated P_YPK1-ΔCBS cells, sphingolipid levels were significantly lower than in GFP-Com2-overexpressing (-Dox) cells (Figure 5H, lane 8 vs. lane 10), but only slightly reduced compared with wild-type (Figure 5H, lane 6 vs. lane 8), consistent with the intermediate Myr sensitivity of P_YPK1-ΔCBS cells (Figure 5B).

Notably, the double mutant P_YPK1-ΔCBS and P_LCB1-ΔCBS (in which the CBS sequence was deleted from the LCB1 promoter) did not exhibit Myr sensitivity comparable to that of com2Δ (Figure 5—figure supplement A-C).

Taken together, these results suggest that Ypk1 represent a major Com2 target under sphingolipid-depleted conditions, although additional Com2-dependent targets likely contribute to sphingolipid homeostasis.

Mapping COM2 regulatory regions required for sphingolipid-dependent Com2 expression

To identify the regions required for Com2 expression control, we constructed a series of plasmids carrying stepwise deletions in either the ∼500 bp 5′ upstream region (putative COM2 promoter) or the COM2 ORF (Figure 6A). These plasmids were introduced into com2Δ cells, and we compared Myr sensitivity and Com2 protein abundance in the presence or absence of Myr (Figure 6). Whereas com2Δ cells were Myr-sensitive, introduction of most plasmids restored Myr resistance to varying degrees (Figure 6B). In contrast, the Δ2-190 mutant and truncation mutants lacking the DNA region corresponding to the Zn-finger domain (1-379, 1-230, 1-143, 1-87) failed to complement Myr sensitivity.

Figure 6.

We next examined the expression of these mutants by immunoblotting. For the promoter-deletion series, removal of ≥180 bp from the 5′ upstream region caused a marked reduction in basal Com2 abundance. Nevertheless, all promoter-deletion constructs still showed a clear increase in Com2 levels upon Myr treatment (Figure 6C). Consistently, in a strain in which the endogenous COM2 promoter was replaced with a tet-off promoter (P_tet-off-13Myc-COM2), Com2 levels increased over time after Myr addition both under “induced (-Dox)” and “repressed (+Dox)” conditions (Figure 6—supplement 1). These results suggest that Myr-dependent upregulation of Com2 is not primarily driven by the native promoter.

In contrast, stepwise deletions within the COM2 ORF revealed a prominent effect on Com2 abundance. Deletion of ≥40 amino acids from the N-terminus caused a strong accumulation of Com2 protein irrespective of Myr treatment (Figure 6D). A C-terminal truncation removing 213 residues (1-230) still exhibited Myr-dependent induction, whereas more extensive C-terminal truncations (1-143 and 1-87) accumulated to high levels regardless of Myr (Figure 6E). Together, these findings indicate that Com2 expression control is largely determined by sequences within the ORF, rather than by the promoter.

Com2 is rapidly degraded by the ubiquitin-proteasome system in response to increased sphingolipid levels

Because Com2 abundance increases upon Myr-induced sphingolipid depletion and this response is independent of the promoter, we next asked whether increasing intracellular sphingolipids would conversely downregulate Com2. Three hours after Myr treatment, we added phytosphingosine (PHS), a ceramide precursor, to bypass the blocked step and restore sphingolipid synthesis (Figure 7A). Com2 levels, which were elevated by Myr, dropped rapidly within 1 hr of PHS addition (Figure 7B, C). In contrast, Ypk1 levels also increased upon Myr treatment but did not show a significant decrease after PHS addition (Figure 7B, D). Notably, TORC2-dependent phosphorylation of Ypk1 (p-Ypk1^T662) increased over time during Myr treatment and declined promptly after PHS addition (Figure 7B, E). These results suggest that Com2 abundance is dynamically tuned to intracellular sphingolipid levels.

Figure 7.

To pinpoint the biosynthetic step responsible for the PHS-dependent reduction in Com2, we used loss-of-function mutants and step-specific inhibitors targeting distinct stages of sphingolipid synthesis (Figure 7A). Blocking mannnosyldiinositol phosphorylceramide, M(IP) ₂C synthesis (ipt1Δ) did not impair the PHS-dependent decrease in Com2, which remained comparable to that in wild-type cells (Figure 7F, G). By contrast, inhibiting ceramide synthesis (lip1-1 +Dox), IPC synthesis (aur1-19 +AbA), or mannnosylinositol phosphrylceramide, MIPC synthesis (csg2Δ) abolished the PHS-dependent reduction in Com2 (Figure 7F, G). Thus, Com2 downregulation requires flux through the pathway up to MIPC. The rapid PHS-induced change in Com2 expression correlated with changes in p-Ypk1^T662 levels in wild-type cells and in mutants impaired at different steps of sphingolipid metabolism, except for those in csg2Δ cells (Figure 7F, H).

We next considered that the rapid loss of Com2 following PHS addition might reflect regulated protein degradation, such as proteasomal degradation or autophagy. Co-treatment with the proteasome inhibitors MG132 and bortezomib markedly suppressed the PHS-dependent decrease in Com2 and led to the appearance of high-molecular-weight species consistent with polyubiquitinated Com2 (Figure 7I, J). In contrast, deletion of ATG5, which is essential for autophagosome formation, did not prevent the PHS-dependent decrease in Com2 (Figure 7—figure supplement). Together, these data indicate that Com2 is downregulated primarily through ubiquitin-proteasome system (UPS)-dependent degradation.

In summary, our results support a model in which an increase in the complex sphingolipids triggers UPS-dependent degradation of Com2, thereby providing negative feedback to restrain downstream sphingolipid synthesis (Figure 7K).

Identification of Com2 ubiquitination sites and the role of phosphorylation in sphingolipid-dependent Com2 degradation

Because deletion of amino acids 2-40 markedly increased Com2 expression levels (Figure 6D), we hypothesized that this region is involved in PHS-dependent degradation. Consistent with this idea, PHS-dependent degradation of Com2 was completely abolished in the mutant lacking amino acids 2-40 (Figure 8A).

Figure 8.

To identify ubiquitination sites, lysine residues located within or near this region were substituted with arginine (Figure 8B). Mutation of K23 and K35, either individually or together, or mutation of K51 alone, did not affect PHS-dependent degradation. In contrast, simultaneous mutation of K23, K35, and K51 (KR mutant) significantly suppressed PHS-induced degradation (Figure 8C-E). The KR mutant fully complemented the Myr sensitivity of the com2Δ strain, indicating that these substitutions do not impair Com2 function (Figure 8—figure supplement 1A). Notably, the KR mutant exhibited a PHS-induced mobility shift that was reversed by λphosphatase (λPPase) treatment, indicating phosphorylation-dependent modification (Figure 8F). These results suggest that ubiquitination at K23, K35, and K51 promotes proteasome-dependent degradation of Com2.

Because degradation was suppressed in the KR mutant despite increased phosphorylation, we next examined the role of phosphorylation in Com2 stability. Phos-tag SDS-PAGE resolved several phosphorylated Com2 species that shifted downward following λPPase treatment, demonstrating that Com2 is highly phosphorylated (Figure 8G, lanes 1, 2).

In addition to the five phosphorylation sites annotated in the Saccharomyces Genome Database, we identified five predicted AGC kinase target sites, including putative Ypk1 phosphorylation sites, yielding a total of ten candidate phosphorylation sites (Fig. 8—figure supplement 1B). Progressive substitution of these serine/threonine residues with alanine (ST-A3 to ST-A10) caused stepwise reduction of phosphorylation-dependent band shifts, with ST-A10 showing a marked loss of phosphorylation (Figure 8G, lanes 2-7).

Consistent with this result, PHS-dependent degradation was unchanged in ST-A3 but progressively suppressed in mutants containing five or more substitutions and was significantly reduced in ST-A10 (Figure 8H-J). Together, these results indicate that phosphorylation of Com2 is required for its PHS-dependent degradation.

Discussion

Bilayer assembly during membrane biogenesis requires the coordinated supply and organization of multiple lipid species, necessitating integrated control of lipid synthesis, uptake, metabolism, and subcellular distribution (Nohturfft and Zhang, 2009). Global regulation of lipid metabolism is achieved not only through signaling pathways that modulate the activity of lipid metabolic enzymes, but also through master transcriptional regulators that coordinate expression of genes encoding these enzymes and their modulators. Master transcriptional regulators have been identified for glycerophospholipids, lipid-saturation control, and sterol metabolism in both yeast and mammals (Covino et al., 2018; Nohturfft and Zhang, 2009). In contrast, although sphingolipid metabolic flux is well understood to be regulated through the TORC2-Ypk1 pathway via control of biosynthetic enzyme activity (Berchtold et al., 2012), transcriptional mechanisms governing sphingolipid metabolism remain far less defined (Breslow and Weissman, 2010).

In this study, we show that Com2 functions as a master transcriptional regulator of sphingolipid metabolism in budding yeast by responding to reduced sphingolipid levels, upregulating its own expression, and promoting Ypk1 expression to stimulate sphingolipid synthesis.

Previous work reported that com2Δ cells are hypersensitive to sulfur dioxide (SO₂) and that Com2 acts as a major transcription factor controlling expression of genes required for SO₂ tolerance (Lage et al., 2019). Com2 has also been identified as a regulator of the UASru element within the promoter of IME1, the master regulator of meiosis in budding yeast, where deletion of this element markedly reduces IME1 transcription (Kahana-Edwin et al., 2013). How the Com2-dependent program uncovered here intersects with these pathways remains unclear; however, Com2 may employ shared regulatory logic across distinct physiological responses, an idea that will be important to address in future studies.

YPK1 is a major Com2 target, but additional targets likely contribute to sphingolipid homeostasis

Our data show that YPK1 is one of the major transcriptional targets of Com2 in sphingolipid metabolism. A genome-wide search based on the Com2-binding site (CBS) consensus sequence also identified a CBS-like element in the promoter of LCB1, which encodes a catalytic subunit of SPT, and a LacZ reporter assay further suggested that the LCB1 promoter can respond to Com2. Notably, deletion of the CBS from the YPK1 promoter (P_YPK1-ΔCBS) reduced Myr-induced Ypk1 upregulation to a level comparable to that seen in com2Δ cells (Figure 5D). However, this promoter mutant did not phenocopy the strong Myr sensitivity of com2Δ. Likewise, a double mutant lacking CBS elements in both the YPK1 and LCB1 promoters failed to recapitulate the com2Δ phenotype (Figure 5—figure supplement). Together, these results suggest that, in addition to YPK1 and LCB1, Com2 likely controls other target genes that collectively contribute to sphingolipid homeostasis.

Com2 degradation is triggered by a cellular readout of complex sphingolipids

Com2 levels increased when sphingolipid synthesis was reduced by Myr treatment, whereas exogenous addition of phytosphingosine (PHS), a ceramide precursor, rapidly promoted Com2 degradation via the ubiquitin-proteasome system (UPS) (Figure 7B, I). Systematic inhibition of individual steps in the sphingolipid pathway further suggested that the PHS-dependent degradation signal reflects the status of complex sphingolipids at or downstream of MIPC (Figure 7F). In glycerophospholipid and sterol metabolism, several lipid-sensing transcriptional systems are well established—for example, Opi1 sensing phosphatidic acid (Loewen et al., 2004), Mga2/Spt23 sensing membrane lipid saturation to control OLE1 expression (Ballweg et al., 2020; Romanauska and Köhler, 2021), and Upc2 sensing sterol depletion to induce sterol biosynthetic genes (Yang et al., 2015). In contrast, the molecular mechanisms by which cells sense sphingolipid levels, particularly complex sphingolipids, remain poorly understood (Breslow and Weissman, 2010). TORC2-Ypk1 signaling is also thought to be influenced by complex sphingolipid status (Figure 7F, H), yet the identity of the upstream sensor is still unclear. Thus, dissecting the mechanism of Com2 degradation may provide a tractable entry point to uncover the elusive sphingolipid sensor operating at the top of this pathway.

How are Com2 ubiquitination and degradation controlled?

Many transcription factors are regulated through proteolysis by the UPS in response to environmental and metabolic cues, including oxidative stress, heat shock, nutrient limitation, changes in cellular redox status, and shifts in metabolic state (e.g., glucose availability, AMP/ATP, NAD⁺/NADH, and TCA-cycle metabolites) (Geng et al., 2012). Within the UPS, E3 ubiquitin ligases act as central decision-makers that determine whether a substrate is degraded. In many cases, this decision is implemented through the recognition of degron motifs and their modulation by post-translational modifications, thereby converting stress/metabolic signals into changes in protein half-life (Finley et al., 2012).

Here, we found that PHS-induced degradation of Com2 critically depends on its N-terminal region (notably the segment around residues 2-40) (Figure 8A). Moreover, substitution of lysine residues near the N terminus with arginine (K23R, K35R, and K51R; hereafter the KR mutant) markedly attenuated PHS-dependent Com2 degradation. These observations suggest that the N-terminal region may provide ubiquitin-acceptor lysines and/or serve as a degron that is recognized by an E3 ubiquitin ligase.

Notably, the KR mutant displayed an increased phosphorylation signal after PHS treatment (Figure 8F). In budding yeast, the transcription factor Gcn4 is phosphorylated by Pho85 under non-starvation conditions, which generates a phosphodegron that is recognized by SCF^Cdc4, leading to ubiquitination and proteasomal degradation (Meimoun et al., 2000). By analogy, Com2 phosphorylation may similarly act as a trigger for UPS-dependent degradation, potentially by creating or exposing a phosphodegron. Consistent with this idea, a mutant in which multiple predicted phosphorylation sites were collectively replaced (ST-A10) exhibited reduced phosphorylation and a significant suppression of PHS-dependent degradation (Figure 8G-J). Together, these results support a model in which Com2 phosphorylation is an important upstream step in its degradation. Nevertheless, the increased phosphorylation observed in the KR mutant could also reflect accumulation of phosphorylated Com2 due to impaired turnover, and future work will be required to establish the causal order of phosphorylation and ubiquitination.

Based on these findings, we propose the model shown in Figure 9. When intracellular sphingolipid levels are low, Com2 is stabilized and promotes sphingolipid synthesis by upregulating genes that positively regulate the pathway and contain Com2-binding sites (CBS), such as YPK1 and LCB1. Conversely, when sphingolipid levels are high, an as-yet-unidentified complex sphingolipid sensor and downstream signaling pathway may promote Com2 phosphorylation, recruit an E3 ubiquitin ligase, and drive Com2 ubiquitination followed by rapid proteasomal degradation.

Figure 9.

Is transcriptional control of sphingolipid metabolism evolutionarily conserved?

Whether a Com2-like mechanism of sphingolipid metabolic control is evolutionarily conserved in mammals remains an open question. In mice, ZFP750 (ZNF750 in humans) has been reported to function as a master regulator of ceramide synthase genes, particularly in the skin, where it is essential for establishing epidermal barrier function by maintaining proper lipid composition (Butera et al., 2023). ZFP750 directly binds to the promoters of lipid metabolic enzyme genes, including Elovl6 and Elovl7, which are involved in fatty acid elongation, thereby regulating lipid metabolism at the transcriptional level.

Although Com2 cannot be considered a clear ortholog of ZFP750, it is tempting to speculate that ZFP750 may also be subject to functional regulation linked to sphingolipid levels, analogous to that observed for Com2. Notably, more than 700 C2H2-type zinc finger proteins are annotated in the human genome (Nabeel-Shah et al., 2024), raising the possibility that additional transcription factors may regulate sphingolipid metabolism in a manner similar to Com2.

Our findings therefore provide a conceptual framework for understanding sphingolipid homeostasis as a transcriptionally controlled process that may be conserved beyond yeast.

Materials and Methods

Strains, plasmids, and media

Descriptions of the strains and plasmids used in this study are presented in Tables 2 and Table 3. Escherichia coli DH5α or JM109 was used as the bacterial host for plasmid construction. Coding sequences were amplified by means of PCR, using KOD-Plus-Neo polymerase (Toyobo) or PrimeSTAR^® GXL polymerase (TaKaRa Bio Inc.). The plasmids were sequenced to ensure that no mutations were introduced owing to the manipulation. Mutant constructs were generated using site-directed mutagenesis and confirmed using sequencing. The media used for yeast culture were YPD (1% yeast extract, 2% peptone, and 2% glucose) or synthetic dextrose (SD) medium (2% glucose and 0.67% yeast nitrogen base without amino acids). Appropriate amino acids and bases were added to the SD medium, as necessary. Yeast cells were cultured at 26°C, unless otherwise stated. All deletion and tagged strains were constructed using homologous recombination with PCR-generated DNA fragments, as described previously (Longtine et al., 1998). Multiple-mutant strains of S. cerevisiae were generated by genetic crossing of the respective single-mutant strains followed by tetrad dissection analysis.

Table 2.

Table 3.

Yeast growth assays

For the yeast plate growth assay, cells were adjusted to an OD₆₀₀ of 1.0, serially diluted in fourfold steps, and 5 μL of each dilution was spotted onto assay plates. Plates were incubated at the indicated temperature for 3-4 days and then photographed.

For the liquid growth assay, as described previously (Ishino et al., 2022), cells were inoculated into YPD medium or YPD supplemented with Myr at an initial OD₆₀₀ of 0.1. Growth was monitored every 30 min at 26°C using a microplate reader (Tecan Sunrise, Tecan Japan, Co., Ltd, Kanagawa, Japan) with three biological replicates.

Antibody production

The rabbit polyclonal antibody that recognizes Com2 was produced as follows. A His₆-fused Com2 protein was expressed in E. coli Rosetta-gami B (DE3) pLysS cells and the total cell lysate was separated on an SDS-PAGE gel. The band corresponding to the His₆-Com2 fusion protein was excised from the SDS-PAGE gel and electroeluted from the gel slices using an AE-3590 electrochamber (Atto). An aliquot of approximately 300 μg of the fusion protein was emulsified with Freund’s complete adjuvant and injected intramuscularly or subcutaneously into young female Japanese white rabbits. Antisera were purified using an affinity column immobilized with His₆-ProS2-Com2 (382-443 a.a.) fusion protein.

Western blot

Protein extracts were prepared using the TCA precipitation method, loaded onto normal or Phos-tag SDS-PAGE gels, and transferred to PVDF membranes (Fujifilm Wako). λPPase treatment was performed as described previously (Ishino et al., 2022). The antibodies and dilutions used in this study were listed in Table 4.

Table 4.

Sphingolipid analysis

Sphingolipids were extracted from S. cerevisiae and analyzed using TLC, as described previously (Yamaguchi et al., 2018).

Construction of LacZ reporter plasmids

The fragments containing the promoter regions of YPK1 and LCB1 were amplified by means of PCR and cloned into the XhoI and BamHI sites of the 4×CDRE-LacZ fusion plasmid, pAMS366 (Stathopoulos and Cyert, 1997) (gifted by Dr. Martha Cyert, Stanford University) using the In-Fusion^® Cloning Kit (Clontech), resulting in pP_YPK1-LacZ and pP_LCB1-LacZ reporter plasmids, respectively.

Genome editing for generation of the P_YPK1-ΔCBS strain

The pMT1447 plasmid, constructed from the pSpCas9(BB)-2A-GFP plasmid (Ran et al., 2013) combined with the Z₄EV expression system (McIsaac et al., 2013), co-expresses the nuclease protein Cas9 and gRNA, which guides the Cas9 protein with the target promoter sequence of YPK1. The repair DNA fragment was amplified from pNK19 carrying the P_YPK1-ΔCBS fragment. The yeast wild-type strain was co-transformed with the pMT1603 plasmid and the repair DNA fragment using the lithium acetate transformation method. After selecting the uracil prototrophic transformants, the genomic DNA purified from the yeast transformants was used for genotyping by means of PCR using specific primers, to confirm the deletion of the CBS in the chromosomal YPK1 promoter. The pMT1603 plasmid was lost by means of 5-FOA counter selection.

Statistical analysis

Statistical analysis was performed using Prism 10 (GraphPad) or Excel (Microsoft) software, and the specific tests carried out have been mentioned in the text and figure legends.

Data availability

All data needed to evaluate the conclusions in the paper are present in the main text and/or the supplementary materials.

Acknowledgements

We express our deep appreciation to Prof. Scott D. Emr for generously providing the plasmids and strains. We thank Profs. Isamu Kameshita and Noriyuki Sueyoshi for providing the λPPase-expressing plasmid. We also thank Prof. Martha Cyert for providing the LacZ reporter plasmid, pAMS366. We also appreciate Prof. Takayuki Sekito for his technical advice on the experimental methods. We acknowledge the technical expertise of the DNA Core Facility of the Gene Research Center, Kagawa University. This work was supported by JSPS KAKENHI grant numbers JP19K05828, 23K26835 (to M.Tabuchi).

Additional information

Author contributions

Mitsuaki Tabuchi designed the research; Kosei Matsumoto, Ayane Nagai, Nao Komatsu, Yuko Ishino, Rina Shirai, Toshiya Ueno, Mio Masaki, and Mitsuaki Tabuchi performed the research; Kosei Matsumoto, Ayane Nagai, Nao Komatsu, Yuko Ishino, Rina Shirai, and Mitsuaki Tabuchi analyzed the data; Kosei Matsumoto, Ayane Nagai, and Mitsuaki Tabuchi wrote the manuscript; Motohiro Tani, Tatsuya Maeda, Naotaka Tanaka, and Ken-taro Sakata reviewed and edited the manuscript; Tatsuya Maeda provided resources; and Mitsuaki Tabuchi supervised the study and was responsible for funding acquisition and project administration.

Funding

MEXT | Japan Society for the Promotion of Science (JSPS) (JP19K05828)

• Mitsuaki Tabuchi

MEXT | Japan Society for the Promotion of Science (JSPS) (23K26835)

• Mitsuaki Tabuchi

Additional files

Figure 1—figure supplement 1. Screening to identify sphingolipid metabolism related factors that suppress Myriocin sensitivity of lip1-1 cells. Wild-type cells and lip1-1 cells carrying a plasmid including each suppresser gene were spotted in 10-fold serial dilution on YPD supplemented with 0.2 µM Myriocin (Myr) in the presence of Dox (20 µg/mL).

Figure 1—figure supplement 2. The diploid homozygous knockout cells of MLM genes were spotted at a 10-fold serial dilution on YPD supplemented with 0, 1.0, 2.0 µM Myr and incubated at 26°C for 2-3 days.

Figure 1—figure supplement 3. (A) Wild-type, ypk1Δ, com2Δ, hog1Δ, slt2Δ and ire1Δ cells were spotted in 10-fold serial dilution on YPD for each condition (26°C, 37°C, 75 mM Acetic acid (AcOH), 50 mM Tris-HCl pH 8.0, 1 M NaCl,0.25 μg/mL Tunicamycin (TN), 20 μg/mL Calcofluor white (CFW), 0.0150% SDS, 50 ng/mL Miconazole, 0.6 μM / 1.0 μM Myr, and 75 ng/mL AbA. (B) Detailed description of each step of the de novo sterol biosynthetic pathway in the yeast Saccharomyces cerevisiae and its experimental mechanism of action. The pathway and proteins responsible for the synthesis of yeast sterols are shown. (C) The indicated cells were spotted in 10-fold serial dilutions onto YPD supplemented with 10 µM, 50 µM, and 100 µM Terbinafine. (D, E) Wild-type cells were grown to mid-log phase in SD liquid medium and separately treated with 1.0 µM Myr, 50 µM Terbinafine, or 50 ng/ml Miconazole for 3 hrs at 26°C, after which lysates were separated by SDS-PAGE and immunoblotted with anti26 Myc or anti-G6PDH antibodies to detect Com2-13Myc or G6PDH (loading control), respectively. The amount of Com2-13Myc in wild-type cells (without Myr) was normalized to the amount of G6PDH, and each data was set as 100%. 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<0.0001, ns: not significant).

Figure 4—figure supplement 1. In silico analysis to identify the candidate genes of the Com2 dependent expression in response to reduced sphingolipids. Schematic representation of the workflow for the bioinformatic approach towards identifying Com2 targets from the S. cerevisiae genome using Yeast Genome Pattern Matching (https://www.yeastgenome.org/nph38-patmatch).

Figure 4—figure supplement 2. Raw immunoblot data used for the quantification shown in Figure 4E. P_tet-off-GFP-COM2 cells were grown to mid42 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 from six independent experiments were subjected to SDS-PAGE followed by immunoblotting with anti-Ypk1, anti-Myc, or anti-G6PDH antibodies to detect Ypk1, Lcb1-13Myc, or G6PDH (loading control), respectively.

Figure 5—figure supplement 1. Evaluation of myriocin sensitivity in YPK1 and LCB1 promoter CBS-deletion mutants. (A) Wild-type, com2Δ, P_YPK1-ΔCBS, P_LCB1-ΔCBS, or P_YPK1-ΔCBS/P_LCB1-ΔCBS double-mutant strains were spotted as 10-fold serial dilutions (starting at OD600=1) onto YPD agar plates containing myriocin (0, 0.6, or 0.8 μM) and incubated at 26°C for 2-3 days to assess myriocin sensitivity. (B) The same strains were cultured in YPD liquid medium containing myriocin (0 or 0.4 μM) in microplates, and growth curves were obtained by measuring OD600 every 30 min. (C) OD_600nm values at 20 hrs of cultivation in the presence of myriocin (0 or 0.4 μM) were quantified (n=3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (**p<0.01, ****p<0.0001, ns: no significant difference).

Figure 6—figure supplement 1. Myriocin-dependent upregulation of Com2 expression is independent of the promoter. P_tet-off-13Myc-COM2 cells were grown to logarithmic phase in SD liquid medium in the presence (+Dox) or absence (−Dox) of doxycycline. Myriocin was then added to a final concentration of 1 μM, and protein extracts were prepared at the indicated time points. The expression levels of 13Myc-Com2 and Ypk1 were analyzed by immunoblotting. G6PDH was used as a loading control.

Figure 7—figure supplement 1. Wild-type and atg5Δ cells expressing Com2-13Myc were treated with 1.0 µM Myr for 3 hrs., followed by the addition of 10 µM PHS and further incubation for 3 hrs. Samples were collected at the indicated time points, and Com2-13Myc protein levels were analyzed by immunoblotting. G6PDH was used as a loading control.

Figure 8—figure supplement 1. (A) com2Δ cells expressing wild-type Com2, an empty vector, or Com2 mutants carrying substitutions at the predicted ubiquitination sites were spotted in 10-fold serial dilutions onto YPD medium containing the indicated concentrations of myriocin (Myr). (B) Analysis of putative phosphorylation sites in Com2. Phosphorylation sites of Com2 annotated in the Saccharomyces Genome Database (SGD), putative Ypk1 phosphorylation sites reported by Muir et al., and AGC kinase–dependent phosphorylation sites in Com2 predicted using the GPS web server (http://gps.biocuckoo.org/online.php) are shown. Serine/threonine residues mutated to alanine in the stepwise phosphorylation-site mutants are indicated. (C) Wild-type or com2Δ cells transformed with plasmids expressing wild-type Com2 or the indicated phosphorylation-site mutants were spotted in 10-fold serial dilutions onto YPD medium containing the indicated concentrations of Myr.

Source data 1

Source data 2