Regulation of sphingolipid synthesis by the C2H2 zinc finger transcription factor Com2 through ubiquitin-proteasome mediated degradation pathway

  1. Kosei Matsumoto
  2. Ayane Nagai
  3. Nao Komatsu
  4. Yuko Ishino
  5. Rina Shirai
  6. Toshiya Ueno
  7. Mio Masaki
  8. Ken-taro Sakata
  9. Motohiro Tani
  10. Tatsuya Maeda
  11. Naotaka Tanaka
  12. Mitsuaki Tabuchi

  • Curated by eLife

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    eLife Assessment

    This study provides valuable insights into how cells maintain sphingolipid homeostasis through transcriptional control and regulated protein degradation in response to changes in sphingolipid levels. The evidence supporting the conclusions is convincing overall, with solid genetic and biochemical approaches, while some mechanistic aspects remain to be clarified. This work will be of interest to researchers studying lipid metabolism and membrane biology.

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Abstract

Membrane lipid synthesis is globally coordinated by a limited set of master transcription factors that regulate broad gene networks encoding lipid-metabolic enzymes and their regulators. Here, we identify the C2H2 zinc-finger transcription factor Com2 as a regulator of sphingolipid homeostasis in Saccharomyces cerevisiae that promotes transcription of downstream targets, including the protein kinase Ypk1, a key activator of sphingolipid synthesis. Com2 protein abundance increased upon treatment with myriocin, an inhibitor of sphingolipid synthesis, but rapidly decreased after addition of phytosphingosine (PHS), a precursor of complex sphingolipids; this decrease was blocked by proteasome inhibitors. These results suggest that Com2 is regulated in a sphingolipid-dependent manner through proteasome-mediated degradation. Moreover, a Com2 mutant in which lysine residues putatively involved in ubiquitination were replaced with arginine exhibited attenuated PHS-dependent degradation and elevated phosphorylation. Likewise, a mutant in which putative phosphorylation sites were replaced with alanine showed reduced PHS-dependent degradation. Together, these findings indicate that Com2 undergoes phosphorylation-dependent degradation via the ubiquitin–proteasome system in response to sphingolipid levels.

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  1. eLife

    eLife Assessment

    This study provides valuable insights into how cells maintain sphingolipid homeostasis through transcriptional control and regulated protein degradation in response to changes in sphingolipid levels. The evidence supporting the conclusions is convincing overall, with solid genetic and biochemical approaches, while some mechanistic aspects remain to be clarified. This work will be of interest to researchers studying lipid metabolism and membrane biology.

  2. eLife

    Reviewer #1 (Public review):

    Matsumoto et al. identify Com2, a C2H2-type zinc finger transcription factor not previously linked to sphingolipid metabolism, as a regulator of this pathway in budding yeast. They show that depletion of sphingolipids by myriocin, an inhibitor of serine palmitoyl transferase, increases Com2 expression. This, in turn, promotes the expression of the protein kinase Ypk1 and enhances TORC2-dependent phosphorylation of Ypk1. The authors identify a Com2-binding site in the YPK1 promoter and provide evidence that Com2 functions upstream of Ypk1 to regulate its
    expression. They further report that Com2 abundance is controlled by the ubiquitin-proteasome system: degradation of Com2 is inhibited by myriocin treatment and enhanced by phytosphingosine. Mutational analyses of putative phosphorylation and ubiquitination sites support a role for these modifications in regulating Com2 stability. Based on these findings, the authors propose that Com2 acts as a transcriptional regulator of sphingolipid metabolism that responds to sphingolipid levels and promotes Ypk1 expression.

    Strengths:

    This study provides a valuable finding on the regulation of sphingolipid synthesis by the transcription factor Com2 in budding yeast. The evidence supporting the authors' claims is solid, although additional evidence clarifying the mechanisms and biological significance of ubiquitin-proteasome-mediated degradation of Com2 would strengthen the work. This work will be of interest to microbiologists studying budding yeast.

    Weaknesses:

    The biological significance of Com2 degradation is not sufficiently clear, which represents an important limitation of the study. It would also be important to determine whether Com2 is actively degraded under normal growth conditions, such as during logarithmic growth in the absence of drug treatment.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    In this study, Matsumoto and co-workers use budding yeast as a model organism to identify and characterize transcriptional mechanisms that homeostatically regulate sphingolipid metabolism. Through a genetic suppressor screen and a series of genetic, molecular, and biochemical analyses, they identify the transcription factor Com2 as a key regulator that responds to sphingolipid levels and regulates the expression of genes such as YPK1, which in turn controls the activity of several enzymes in the yeast sphingolipid biosynthetic pathway.

    Com2 itself is further regulated by the ubiquitin proteasome system in response to sphingolipid levels. High sphingolipid levels promote proteasomal degradation of Com2, whereas low sphingolipid levels stabilize Com2. These findings suggest that Com2 is a central component of a feedback system that helps maintain sphingolipid homeostasis.

    Strengths:

    The identification of Com2 as an upstream regulator of the TORC2-Ypk1 pathway is supported by multiple orthogonal lines of evidence. The authors also provide mechanistic insight into how Com2 protein levels are dynamically controlled through phosphorylation and ubiquitin-mediated degradation. Stabilization of Com2 in response to sphingolipid depletion appears to be required for the transcriptional upregulation of YPK1 expression.

    Weaknesses:

    Although several important questions remain unresolved, such as which kinases function upstream of Com2 and which ubiquitin ligase(s) target Com2, this work is nevertheless likely to have a meaningful impact on the field of sphingolipid metabolism. The identification of a regulated transcription factor that responds to sphingolipid levels may also be of broader interest to researchers studying membrane homeostasis.

  4. eLife

    Reviewer #3 (Public review):

    This paper extends the authors' 2022 studies of how the synthesis of membrane sphingolipids is regulated in budding yeast. Here, they hypothesized that overexpression of a protein involved in sphingolipid (SL) biosynthesis would confer resistance of lip1-1 cells, which are Dox-inducibly defective in expression of a ceramide synthase regulatory subunit, to myriocin (Myr), a serine palmitoyltransferase inhibitor that inhibits SL synthesis. To test this idea, they transformed lip1-1 cells with a multi-copy genomic library, selecting for Myr resistance. Apart from LIP1 itself and YPK1, a protein kinase downstream of TORC2, COM2, which encodes the Com2 C2H2-type zinc finger transcription factor, was the most frequent hit in the screen. They went on to show that com2Δ cells exhibited Myr sensitivity, and that Com2 protein expression was induced under conditions that reduced complex sphingolipid synthesis, such as Myr-treatment. Using ypk1-as ypk2Δ cells and the 3-MB-PP1 Ypk1as a selective Ypk1as kinase inhibitor, they showed that Com2 phosphorylation was independent of Ypk1 activity, suggesting that Ypk1 lies downstream of Com2. Consistently, Myr treatment, which reduces SL synthesis, resulted in an increase in both Com2 and Ypk1 proteins. By generating a Ptet-off-GFP-COM2 strain they showed that when Dox was removed to induce GFP-Com2 overexpression, Myr resistance was increased. They went on to show that Com2 binds to a Com2 response element in the YPK1 promoter and drives expression of Ypk1. This was confirmed by showing that expression of a YPK1-driven lacZ reporter gene was also elevated when GFP-Com2 overexpression was induced. CRISPR deletion of the putative Com2-binding site (CBS) from the endogenous YPK1 promoter was used to generate PYPK1-ΔCBS cells, which showed a significant reduction in Ypk1 expression and exhibited intermediate Myr sensitivity, suggesting that Com2 is important for but not the only regulator of Ypk1 expression. Analysis of SL levels showed that they largely paralleled the levels of Ypk1 protein and active pT662 Ypk1. Using deletion analysis of the COM2 gene, they showed that residues 2-190 and the C-terminal DNA binding domain of Com2 were essential for Com2 function in the SL synthesis pathway. Deletion of {greater than or equal to}40 amino acids from the N-terminus increased expression of Com2 protein irrespective of Myr treatment, suggesting that Com2 protein levels are regulated by protein stability. Consistently, they found the high level of Com2 protein induced by Myr was rapidly reversed by treatment with phytosphingosine (PHS), a ceramide precursor that bypasses the Myr-blocked step and restores SL synthesis. The reduction in Com2 protein plus PHS was prevented by MG132 proteasome inhibitor treatment and led to the accumulation of polyUb-Com2 species, consistent with Com2 being negatively regulated by SL-induced UPS-mediated degradation. Based on the use of selective inhibitors of different steps in SL synthesis, they showed that SL biosynthesis up to the level of MIPC (mannnosyldiinositol phosphorylceramide) is required for the SL-mediated degradation response. Based on individual and combined K to R mutagenesis of the three Lys in Com2 1-49, they showed that K23, K35 and K51 in combination are needed for PHS-induced Com2 degradation, and therefore are likely to be the main Com2 Ub sites. Finally, they observed that PHS induced an increase in K3R Com2 phosphorylation, finding that an S/T10A mutant was only weakly phosphorylated and was resistant to PHS-induced degradation, suggesting that phosphorylation of Com2 is required for PHS-dependent degradation.

    The paper is clearly written, and the data in Figures 1-6 show convincingly that the Com2 zinc finger protein, by inducing the expression of a set of genes, including YPK1 and LCB1, plays an important role in sphingolipid (SL) homeostasis in yeast under conditions when sphingolipid levels are low. However, the data in Figures 7 and 8, where the authors provide evidence that the Com2 protein was rapidly degraded in a proteasome-dependent manner in response to phytosphingosine (PHS) treatment, dependent on the N-terminal 40 residues of Com2 and a combination of three Lys residues in this region, are intriguing but incomplete. There are a number of issues, including the identity of the Com2 ubiquitylation sites. They showed that the K23/35/51R Com2 mutant was stabilized, but did they provide direct evidence that these three Lys are in fact ubiquitylated (e.g. GG-K peptide enrichment based MS analysis of Ub-Com2 from PHS-treated, MG132-treated cells). They showed that PHS treatment increased Myc13-tagged Com2 ubiquitylation in the presence of MG132, but did not show that the K3R Com2 mutant (or the S/T10A phosphorylation site Com2 mutant) failed to be ubiquitylated. They also found that the WT Com2 and particularly the K3R Com2 mutant protein exhibited hyperphosphorylation in response to PHS treatment, and that mutation of 10 potential pSer sites to Ala abolished this effect, and stabilized the Com2 protein. However, it is unclear whether the K3R mutation led to increased Com2 hyperphosphorylation per se following PHS treatment, or whether this is because there is more K3R protein, as they suggest might be the case. It is also not clear what protein kinase is responsible or how it might be activated when SL levels are high. In addition, the E3 Ub ligase needed for Com2 degradation was not identified, and it is not clear whether Com2 phosphorylation is directly involved in its recognition by a phosphodependent E3 Ub ligase, as they propose in the model shown in Figure 9. Finally, and perhaps most importantly. It is unclear how elevated levels of phytosphingosine or any sphingolipid are sensed by the Com2 pathway in order to switch on the degradation response as a negative feedback event. The model depicted in Figure 9 exposes all of these unknowns. The paper would be significantly strengthened by additional experiments defining how complex SL levels are sensed, how Com2 is phosphorylated in response to SL sensor signals, and how (phospho)Com1 is recognized for ubiquitylation and degradation.

    In summary, the finding that the Com2 zinc finger transcription factor is an upstream regulator of the sphingolipid biosynthesis pathway in budding yeast, acting as part of an SL sensor system to maintain sphingolipid homeostasis, is new and potentially important. However, more mechanistic work needs to be done to address the unanswered questions raised by the data in Figures 7 and 8.

  5. Version published to 10.64898/2026.02.20.707110 on bioRxiv