Yeast lipid droplet dynamics are coupled to sphingolipid biosynthesis via Tsc3p

doi:10.21203/rs.3.rs-2859060/v1

Download PDF

Article

Yeast lipid droplet dynamics are coupled to sphingolipid biosynthesis via Tsc3p

https://doi.org/10.21203/rs.3.rs-2859060/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Regulation of lipid metabolism is pivotal for living cells to maintain the balance between energy production and membrane component assembly. Fatty acids can be stored as triacylglycerols in lipid droplets (LD) or accumulate in various subcellular compartments as diacylglycerols or sphingolipids, which may exert deleterious effects and associate with metabolic diseases. At present, the mechanisms maintaining the balance of neutral lipid synthesis and consumption remain largely unknown. Thus, this study aimed to identify regulators of LD dynamics in yeast.

Based on flow cytometry we assess LD consumption in candidates involved in first steps of sphingolipid synthesis. We found that the Tsc3p deletion strain (tsc3Δ) displayed a decrease in LD consumption, which was reversed by adding phytosphingosine. Assessment of lipidomic profiles revealed decreased ceramide level in tsc3Δ cells. In addition, we determined the subcellular localization of Tsc3p and analogous human small subunits of the serine palmitoyltransferase to the endoplasmic reticulum and that ssSPTb rescues the LD consumption defect in tsc3Δ cells. In conclusion, our data show that tsc3Δ impairs LD breakdown accompanied by low cellular sphingolipid level which lead to suggestion that Tsc3p is required for efficient sphingolipid biosynthesis for maintaining cellular lipid homeostasis.

Biological sciences/Microbiology

Biological sciences/Biochemistry/Lipidomics

Biological sciences/Biochemistry/Lipids

Biological sciences/Biochemistry/Metabolomics

Eukaryotic cells are able to store energy in LDs in the form of triacylglycerol (TAG) and sterol esters. This is evolutionary conserved from yeast to human [1–6]. As central components of the lipid metabolism, LD safeguard the supply of metabolites for cellular processes and play a role in obesity and related diseases such as type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD) [7, 8]. An elevated TAG level is associated with lipotoxic intermediates such as diacylglycerols (DAG) and ceramides, which can cause insulin resistance, partially via protein kinase C (PKC) activation [9–12]. Therefore, the mechanism of lipid storage and release remains of high interest.

Saccharomyces cerevisiae represents a convenient in vivo yeast model to investigate the mechanisms underlying TAG storage and their link to metabolic diseases at the molecular level. LD number and size vary dynamically, reflecting accumulation in the stationary phase and lipolysis during the lag phase, when the demand for membrane lipids increases during cell growth and division [13, 14]. Moreover, the storage of lipids in LDs protects cells from lipotoxicity by channeling excess non-esterified fatty acids (NEFAs) into LD biogenesis [15–19]. LD production occurs in the endoplasmic reticulum (ER), where DAG and CoA-activated NEFAs are combined by the diacylglycerol-acyltransferases Dga1p and Lro1p to TAG [20–22]. At low concentrations, TAGs are localized between the inner and outer leaflets of the ER membrane, but at higher concentrations emerge from the ER membrane into the cytosol as nascent LD [5, 23–27]. Rapid consumption of the LD ensures that stored TAGs can be utilized for synthesis of membrane lipids and occurs via hydrolysis in times of growth and cell proliferation [28–31], mediated by the TAG lipases Tgl3p, Tgl4p and Tgl5p [32–36]. DAG are intermediates of TAG hydrolysis, which are channeled into the membrane lipid synthesis via the Kennedy pathway or cytidine diphosphate diacylglycerol pathway [37–39]. On the other hand, TAG hydrolysis results in NEFA, which can be utilized for energy production during quiescence or channeled into ceramide synthesis [23, 40, 41] as well as all other membrane lipids. The heterodimer serine palmitoyltransferase (SPT), catalyzes the rate-limiting step of sphingolipid synthesis by condensation of palmitoyl-CoA and serine to 3-keto-dihydrosphingosine [42, 43]. Its catalytic activity consists of Lcb1p and Lcb2p, which are reportedly stimulated by Tsc3p (Temperature-sensitive Suppressors of Calcium Sensitive Growth mutants 3) [44]. Continuing studies show that Tsc3p regulates amino acid substrate selectivity by promoting alanine utilization by SPT to form non-canonical 1-deoxy-3KDS and subsequent synthesis of deoxy-LCB products [45]. It was shown that Tsc3p is responsible for an inhibitory effect of alanine on SPT utilization of serine [45]. In addition, Tsc3p is required for binding of phosphatidylinositol-4-phosphate (PtdIns(4)P) phosphatase Sac1p to SPT [46]. Sac1p, primarily regulates the pool of PtdIns(4)P, thereby modulating substrate supply of phosphoinositol (PI) to Aur1p (phosphatidylinositol:ceramide phosphoinositol transferase in yeast), the inositol phosphotransferase Ipt2 and the regulatory subunit for the MIPC synthase Csg2 in subsequent stages of complex sphingolipid synthesis [47, 48].

Previous studies in yeast demonstrate that sphingolipids, in detail ceramides, influence cell growth by regulating the cell cycle checkpoint Swe1 and the cyclin-dependent kinase Cdc28 upstream of the protein phosphatase 2A (PP2A) [29, 31]. Moreover, Cdc28 may also control the activity of the LD-resident lipase Tgl4 [14]. Thus, hypothetically sphingolipids could regulate LD dynamics in addition to cell cycle progression through the PP2A-Swe1-Cdc28 axis in yeast.

Regulation of LD dynamics is largely unknown. Here we report that Tsc3p is required for efficient LD consumption. Our results indicate that Tsc3p is required for efficient sphingolipid biosynthesis to ensure proper lipolytic activity, cell growth and thereby to maintain overall cellular lipid homeostasis.

Decreased LD consumption in tsc3∆ cells

We previously reported a flow cytometric “genome-wide screen” in S. cerevisiae (covered by 5127 deletion strains) for the identification of regulators of LD dynamics describing functional coupling of LD consumption and vacuole homeostasis [54]. Unexpectedly, mutants responsible for sphingolipid synthesis were shown to affect LD dynamics but this aspect was not further investigated. This study therefore focused on the analysis of identified candidate mutant strains which lack genes related to the sphingolipid synthesis downstream of SPT.

Deletion of Tsc3p, a protein previously described to directly interact with SPT in the first step of sphingolipid biosynthesis, showed defects in LD consumption (Fig. 1A) after dilution stationary phase cells into fresh medium containing cerulenin. Furthermore, defective LD consumption was detectable in sac1Δ, csg2Δ and ipt1Δ cells after growth resumption with equal conditions (Fig. 1A).

To focus on the rate limiting step of sphingolipid biosynthesis, experiments were repeated in the presence of myriocin to inhibit the function of SPT. Myriocin-treated control cells (wt) only reduced their LD pool by 15% whereas untreated control cells reduced their LD pool size by 75% and tsc3Δ cells by 47% (Fig. 1B). To further analyze the effect of a direct disruption of the SPT activity on LD dynamics, we used the auxin-inducible degron system to deplete the heterodimeric SPT-component Lcb1. For this, cells in stationary phase were diluted in fresh medium containing cerulenin and auxin. Depletion of Lcb1-AID induced by auxin results in 28% reduced LD pool size after 5 h of growth compared to untreated OsTIR Lcb1-AID cells (Fig. 1C). These findings suggest that inhibition of sphingolipid synthesis affects the consumption of neutral lipids by dysregulation of the lipolytic activity.

In yeast, Tgl3p represent the major lipase, which is active in presence of LDs and hydrolyzes stored TAGs, thereby providing fatty acids for membrane production [32, 34, 36]. Thus, we overexpressed Tgl3p in tsc3Δ cells to increase lipolysis and overcome potential reduced lipolytic activity in these cells. Flow cytometry analysis of LD consumption showed that increased lipolysis by overexpression of Tgl3p does not affect the LD consumption after 5 h of growth in control cells but rescues the defective LD consumption in tsc3Δ cells (Fig. 1D). Next, we examined LD consumption by Tgl4p, the second major lipase of yeast [33, 34], which is regulated by the cyclin dependent kinase Cdc28 [14]. Of note, Cdc28 activity has been linked to sphingolipid synthesis [29]. It was previous shown that deletion of tgl4 results in decreased mobilization of TAG [33]. Our cytometry measurement indicated similar defective LD consumption in tgl4Δ cells as in cells with compromised sphingolipid synthesis like tsc3Δ cells (Fig. 1E).

Phytosphingosine rescues lipid droplet consumption and growth of tsc3∆ cells

Because Tsc3p is a stimulator of sphingolipid biosynthesis, we analyzed the lipidome of control and tsc3Δ yeast cells to explore the influence of tsc3Δ on cellular lipid homeostasis. The lipidomic data of tsc3Δ cells grown for 5 h in cerulenin containing SC-medium revealed decreased cellular ceramide levels in comparison to wt cells (Fig. 2A). Wt cells showed elevated cellular ceramides up to 123% after 5 h of growth, whereas in tsc3Δ cells ceramide level declined to 61.4%. Moreover, tsc3Δ cells showed a decrease in TAG mobilization of 40% after 5 h dilution into fresh medium, confirming indicated LD consumption defect measured by flow cytometry (Fig. 1B). The wt cells consumed 95% of stored TAGs and DAGs were reduced to 64.3% after 5 h of growth. In contrast, DAG levels in tsc3Δ cells did not change in the same time frame.

To investigate the effect of cellular sphingolipid levels on LD consumption in wt and tsc3Δ cells, we analyzed LD consumption in the presence of the ceramide-precursor phytosphingosine (PHS) (Fig. 2B) [62]. Flow cytometry analysis of tsc3Δ cells in stationary phase diluted in fresh medium containing 10 µg/ml cerulenin and 15 µM PHS indicated an elevated LD consumption of 14.9% after 5 h of growth compared to tsc3Δ cells grown without PHS (Fig. 2C). Fluorescence microscopic analyses with same conditions confirmed the flow cytometry results (Fig. S5).

The flow cytometry measurements also revealed a growth defect of the tsc3Δ cells. Previous studies demonstrated a growth defect of the thermosensitive tsc3Δ cells at temperatures of 37°C [44, 63]. The present study extends this finding to lower temperatures at 30°C. Further monitoring of the growth rates showed that the wt strain reaches the stationary phase at OD₆₀₀ of 6.8 after 14 h, whereas the tsc3Δ strain reaches stationary phase only after 26 h with a lower OD₆₀₀ of 3.2 (Fig. 3A). Addition of PHS rescued the observed growth defect of tsc3Δ cells by reaching the stationary phase at OD₆₀₀ of 6.4.

Moreover, we analyzed the influence of induced Lcb1-AID depletion on growth. Auxin-mediated Lcb1-depletion significantly reduced the growth of an OsTIR Lcb1-AID strain at 26 h after growth resumption, which was reversed by supplementation with PHS (Fig. 3B). In addition, predominant expression of Lcb1-AID protein occurs in the exponential growth phase between two and six hours after growth resumption (Fig. 3C). Auxin-induced degradation resets Lcb1-AID protein level to initially low amount of stationary phase after 4 h of growth (Fig. 3D).

Subcellular localization of the candidate protein Tsc3p

Prior studies demonstrated co-immunoprecipitation of Tsc3p with Lcb1p and Lcb2p [44]. As SPT resides in the ER membrane, active in a multimeric SPOTS complex with Tsc3p, we hypothesized that Tsc3p also localizes at the ER membrane [64, 65].

To assess the subcellular localization, we tagged Tsc3p with a green fluorescent protein (GFP) and integrated it into a pRS415 vector, controlled by a constitutive alcohol dehydrogenase (ADH) promotor (pRS415-ADHpr-Tsc3p-GFP). First, we tested the function of the transgenic Tsc3p-GFP in LD consumption and growth in tsc3Δ cells, by transfection of the vector pRS415-ADHpr-Tsc3p-GFP in both the control and tsc3Δ cells and subsequent growth experiments and flow cytometry-based LD analysis. The constitutively expressed Tsc3p-GFP was able to rescue the growth defect and LD consumption in tsc3Δ cells (Fig. 4A, 4B). Then, laser scanning microscopic investigations with transfected wt cells in stationary phase confirmed the co-localization of Tsc3p-GFP and red fluorescent protein (RFP)-tagged HDEL (ER marker) on the perinuclear and cortical ER (Fig. 4C).

Human small subunits of SPT rescue the defective phenotype of tsc3∆ cells

In human cells, two small subunits of SPT (ssSPTa and ssSPTb) have been reported to stimulate the activity of SPT [44, 63]. As a first step to explore whether the process is conserved in humans, we analyzed growth and LD consumption of tsc3Δ cells overexpressing ssSPTa-GFP or ssSPTb-3HA.

Flow cytometry revealed that after 5 h of lipolysis only the ssSPTb-3HA, but not the ssSPTa-GFP rescues the LD consumption defect in tsc3Δ cells to levels similar to the wt cells (Fig. 5A). Conversely, neither the overexpression of ssSPTa nor ssSPTb increased the LD consumption in wt cells.

Moreover, we observed a rescue effect in growth of the tsc3Δ cells overexpressing 3HA-tagged ssSPTb, which reaches the stationary phase at OD₆₀₀ = 6 within less than 25 h similarly to that of wt cells (Fig. 5B). Of note, ssSPTa-overexpressing tsc3Δ cells reach the stationary phase (OD₆₀₀ = 4.6) after 27 h.

We then hypothesized that ssSPTa and ssSPTb can stimulate the activity of yeast SPT, probably by a co-localization in the ER and interaction with SPT like Tsc3p. To explore the cellular localization of ssSPTs, we overexpressed C-terminal GFP-tagged ssSPTa and ssSPTb in a wt strain together with a vector expressing RFP-ssHDEL as marker for the ER. Fluorescence microscopic analysis showed that both ssSPTa-GFP and ssSPTb-GFP localize on the ER similarly to Tsc3p (Fig. 5C).

This study identified Tsc3p as a regulator of LD dynamics with potential to modulate cellular lipid homeostasis and growth. Flow cytometry-based LD analysis demonstrated that deletion of Tsc3p, a stimulator of sphingolipid synthesis, decreases neutral lipid consumption, ultimately regulating LD dynamics in yeast. This effect is reversible with external supplementation of PHS. Our further findings on the defective LD consumption in myriocin-treated wt cells and cells with auxin induced Lcb1 depletion, hint that interruption in the sphingolipid biosynthesis pathway affects the breakdown of neutral lipids. Our data also confirm a growth defect in tsc3Δ cells, which is reversible through PHS supplementation as shown in previous studies [63]. We extend this observation showing that direct interruption of the first step of sphingolipid synthesis by auxin inducible depletion of Lcb1p also results in a growth defect. It has been previously shown that mutation of Lcb1 (lcb1-100) impairs sphingolipid synthesis, leading to low cellular ceramide levels accompanied with reduced growth [66]. Moreover, a growth defect was detected in a double mutant lac1Δlag1Δ yeast strain, which is known for its decreased Cer synthesis [67]. However, in our OsTIR LCB1-AID strain, PHS reverses auxin-induced growth and LD consumption defect as well, which underlines the functional influence of sphingolipids on growth and LD dynamics. The rescue effect on LD consumption together with the observed reconstitution of growth by supplementation of PHS in this study may lead to the notion that cell cycle progression and LD degradation in tsc3Δ cells are disturbed due to a low level of sphingolipids.

Previous research providing evidence for the impact of sphingolipids on growth on the base of a PP2A-Swe1-Cdc28 pathway. In particular C18:1 phytoceramides have been shown to influence the activity of the regulatory subunit B of the protein phosphatase 2A (PP2A^Cdc55), which in turn leads to inactivation of Swe1p, an important cell cycle checkpoint in yeast that connects cell cycle progression and the sphingolipid metabolism [29, 30, 68, 69]. Swe1p is able to arrest the cell cycle by an inhibitory phosphorylation at tyrosine residue 19 of the major cyclin-dependent kinase Cdc28p, ultimately regulating cell cycle progression [31, 70].

The rescue effect on the observed reconstitution of growth by supplementation of PHS may lead to the notion that cell cycle progression in cells like tsc3Δ mutant or cells with depleted Lcb1p are disturbed due to a low level of sphingolipids. It would be conceivable that a reduced level of ceramides affects the activity of PP2A^Cdc55, which in turn leads to an enhanced activation of the cell cycle checkpoint Swe1p. Swe1p is thus able to arrest the cell cycle by an inhibitory phosphorylation of Cdc28p, ultimately diminish cell cycle progression.

It is conceivable that the PP2A-Swe1-Cdc28 pathway regulated by ceramides is also responsible for the defective LD consumption in sphingolipid synthesis-deficient cells like tsc3 deleted, Lcb1p depleted or myriocin treated cells by downregulation of lipolysis. The yeast TAG lipase Tgl4p is phosphorylated and activated by the cyclin-dependent kinase Cdc28p which provides evidence for a direct connection between regulation of the cell cycle progression and NL breakdown [14]. In detail, Tgl4p is activated via phosphorylation at residues Thr⁶⁷⁵ and Ser⁸⁹⁰ by Cdc28p in time of G1/S transition [14]. Of note, cells with a deletion of the Cdc28p regulated lipase Tgl4p showed a similar defect of LD consumption as tsc3Δ cells. Thus Cdc28p could be a branching point for the influence of sphingolipids regulating growth and lipolysis (Fig. 6). Nevertheless, future experiments are needed to address whether reduced ceramide levels in tsc3Δ cells in fact inhibit the activity of Cdc28p, resulting in a decreased lipolytic activity driven by Tgl4p.

The present study shows that the overexpression of human ssSPTb-3HA is able to rescue growth defect and LD consumption in tsc3Δ cells. Overexpression of human ssSPTa-GFP shows a minor effect on growth without rescue of the LD consumption defect. Nevertheless, we cannot exclude that expression of ssSPTa rescues LD consumption. It can be speculated that in our construct the C-terminal GFP tag (27 kDa), which is a big protein in relation to ssSPTa (8.46 kDa), interferes with activity. Nevertheless, our fluorescence microscopic analysis show that both ssSPTa-GFP and ssSPTb-GFP localize on the cortical ER similarly to the Tsc3p and SPT [63]. Based on these findings, we deem that human ssSPTb stimulates the sphingolipid synthesis as proxy for the missing Tsc3p, probably by a co-localization in the ER and interaction with yeast SPT. These results further leads to suggestion that sphingolipid content in cells influences both growth and LD dynamics in yeast. Evidence is found in previous work in which decreased growth is also a phenotypic hallmark of triple knockout lcb1Δlcb2Δtsc3Δ mutant yeast expressing the human LCB1–LCB2 heterodimer [63, 71]. This mutant strain accumulates less long chain bases due to a reduced activity of SPT. The lcb1Δlcb2Δtsc3Δ cells co-expressing either human ssSPTa or ssSPTb along with human LCB1–LCB2 show an increased SPT activity and reconstitute cellular sphingoid base levels accompanied with improved growth [63, 71].

It remains yet unclear where NEFA, mobilized by the supposedly constitutively active major lipase Tgl3p, are channeled in tsc3Δ cells if not utilized for sphingolipid synthesis [32, 34]. Excessive accumulation of NEFA is lipotoxic for yeast and also for human cells [12, 16, 19, 72]. The tsc3Δ cells could prevent lipotoxicity by a previously presented mechanism, by which NEFA continuously cleaved from TAG are repetitively re-esterified to DAG by Dga1, yielding a futile cycle [50]. Accordingly to the research of Gable that claims tsc3Δ results in decreased activity of SPT [44], disturbance of sphingolipid synthesis in tsc3Δ cells could lead to a futile cycle of fatty acids not utilized for sphingolipid synthesis, which would ultimately suppress LD consumption. The present study shows that PHS rescues LD consumption in tsc3Δ cells. It can be speculated that this rescue effect is caused by entrance of mobilized NEFA into the phytoceramide synthesis. Hence C26 very long chain fatty acid (VLCFA) are condensed with PHS to yield phytoceramide [59, 73], an external addition of PHS could break the futile cycle of fatty acids used for C26 VLCFA elongation in tsc3Δ cells. Besides that, overexpression of Tgl3p rescues LD consumption in tsc3Δ cells, suggests that excess released NEFA are channeled into alternative pathways. Previous work indicated that yeast channels excess NEFA towards membrane phospholipid synthesis, which results in increased levels of phosphatidic acid and DAG [74]. We found elevated DAG levels in tsc3Δ cells compared to wt cells after 5 h growth. However, we did not find increased levels of phospholipids in tsc3Δ cells (S3).

In conclusion, we showed that sphingolipid biosynthesis in the ER, stimulated by Tsc3p, is essential for neutral lipid consumption, ultimately regulating LD dynamics. The rescue effect of PHS indicates downregulated lipolysis and attenuated growth due to low level of sphingolipids in tsc3Δ cells. We suppose a model in which Cdc28p is a branching point for cell cycle progression and Tgl4p driven lipolysis. Nevertheless, we cannot exclude accompanying processes of re-esterification of lipolysis-derived fatty acids into TAG, which will be needed to further explore the potential interplay between LD dynamics and sphingolipid metabolism.

Yeast strains and plasmids

The yeast strains used are genetically identical to a BY4741 (MATa; his3∆1; leu2∆0; met15∆0; ura3∆0) background except strain SEY6210 pRS305_OsTIR_3xFLAG (Table 1). Endogenous sequence-tagging was performed by homologous recombination of an integrative PCR construct [49]. As indicated, non-essential gene knock out strains were used from a deletion library of Open Biosystems (Huntsville, Alabama/USA). The plasmids used are listed in Table 2. To generate plasmids carrying C-terminal GFP tagged versions of ssSPTa and ssSPTb, ssSPTs were amplified from mammalian gene collection (MGC) of human Sequence-Verified cDNA (Dharmacon, Lafayette, Colorado/USA), digested with SmaI/SpeI (New England Biolabs (NEB), Ipswich, Massachusetts/USA) and ligated into SmaI/SpeI sites of pRS415-ADHpr-GFP [50]. For C-terminal 3HA tagged version of ssSPTb, ssSPTb was amplified from MGC human ssSPTb Sequence-Verified cDNA (Dharmacon). The ssSPTb PCR product was digested with SpeI/SmaI and ligated into plasmid pRS415-ADHpr-3HA digested the same way [50]. To generate a plasmid carrying C-terminal GFP-tagged Tsc3, Tsc3 was amplified from genomic DNA, digested with BamHI-HF/XbaI (NEB) and ligated into plasmid pRS415-ADHpr-GFP digested the same way. For plasmid carrying C-terminal 3HA-tagged Tgl3, Tgl3 was amplified from genomic DNA and digested with SpeI /HindIII and ligated into SpeI/HindIII sites of pRS415-ADHpr-3HA. Lcb1 was genomically tagged at the C-terminus using an AID-6HA-pHyg cassette amplified from pBW2744-pHyg AID 71-114-6HA [51]. Lcb1-AID-6HA tagging was integrated by homologous recombination of the PCR product AID-6HA-pHyg into SEY6210 pRS305_OsTIR_3xFLAG background strain [52, 53].

Table 1

Yeast strains used in this study
Strain ID	Genotype	Source
BY4741	MATa; his3∆1 leu2∆0 met15∆0 ura3∆0	Brachmann et al 1998 [75]
BY4741 csg2∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; CSG2::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 ipt1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; IPT1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 lac1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; LAC1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 lag1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; LAG1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 lcb3∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; LCB3::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 lcb4∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; LCB4::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 lcb5∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; LCB5::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 orm1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; ORM1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 orm2∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; ORM2::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 sac1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; SAC1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 scs7∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; SCS7::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 sur1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; SUR1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 sur2∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; SUR2::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 tgl4∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TGL4::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 tsc3∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 ydc1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; YDC1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 ypc1∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; YPC1::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
BY4741 ysr3∆	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; YSR3::kanMX	yeast deletion library - Open Biosystems GE Dharmacon
YDM351	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; pRS415-ADHpr-Dga1-GFP	Markgraf et al 2014 [50]
YDM658	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; pRS415	This study
YDM661	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX pRS415	This study
YDM667	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; pRS415-ADHpr-Tgl3-3HA	This study
YDM726	SEY6210 MAT α; leu2-3,112; ura3-52; his3-Δ200; trp1-Δ901; suc2-Δ9; lys2-801; GAL LEU2::pRS305_OsTIR-3xFLAG	Robinson et al. 1988 [53], Serbyn et al. 2020 [52]
YDM728	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX pRS415-ADHpr-Tsc3-GFP	This study
YDM731	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX pRS415-ADHpr-Dga1-GFP	This study
YDM737	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX pRS415-ADHpr-Tgl3-3HA	This study
YDM745	SEY6210 MAT α; leu2-3,112; ura3-52; his3-Δ200; trp1-Δ901; suc2-Δ9; lys2-801; GAL LEU2::pRS305_OsTIR-3xFLAG; Lcb1-AID-6HA	This study
YDM768	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; pRS415-ADHpr-ssSPTb-3HA	This study
YDM770	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX pRS415-ADHpr-ssSPTb-3HA	This study
YDM771	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; pRS415-ADHpr-ssSPTa-GFP	This study
YDM772	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TSC3::kanMX pRS415-ADHpr-ssSPTa-GFP	This study
YDM773	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TRP1::HygB pRS415-ADHpr-ssSPTa-GFP + ss-RFP-HDEL	This study
YDM774	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TRP1::HygB pRS415-ADHpr-ssSPTb-GFP + ss-RFP-HDEL	This study
YDM777	MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; TRP1::HygB pRS415-ADHpr-Tsc3-GFP + ss-RFP-HDEL	This study

Table 2

Plasmids used in this study
Plasmid ID	Description	Source
133	pRS415-ADHpr	Nguyen et al. 2012 [76]
87	ss-RFP-HDEL	Prinz et al. 2000 [77]
185	pRS415-ADHpr-Dga1-GFP	Markgraf et al. 2014 [50]
277	pRS415-ADHpr-Tgl3-3HA	This study
278	pRS415-ADHpr-Tsc3-GFP	This study
309	pRS415-ADHpr-ssSPTb-3HA	This study
310	pRS415-ADHpr-ssSPTa-GFP	This study
312	pRS415-ADHpr-ssSPTb-GFP	This study

Individual strains were grown at 30°C shaking at 180 rounds per minute (RPM) in synthetic complete (SC) growth medium containing 6.7 g/L yeast nitrogen ammonium sulfate (YNB from MP Biomedicals, Irvine, California/USA), 20 g/L glucose and an appropriate addition mixture of amino acids (Complete Supplement Mixture [CSM] 0.79 g/L; CSM-leucine 0.69 g/L; CSM-leucine-tryptophan 0.64 g/L; CSM-histidine 0.77 g/L purchased from MP Biomedicals, Irvine, California/USA).

Assessment of growth curve

Precultured strains in stationary phase were diluted to an optical density at 600 nm wavelength (OD₆₀₀) of 0.5 in appropriate fresh SC medium to induce growth. Growth was detected by OD₆₀₀ measurements at 2-h intervals until the stationary phase was reached using Multi-function plate reader Infinite Pro 200 (TECAN, Maennedorf, Zurich/Switzerland) [54].

Assessment of Lcb1 degradation by Auxin-inducible degron system

The auxin-inducible degron (AID) system is a method to control the cellular protein level and therefore it is useful to study the function of essential proteins in vivo. Through induction by the plant hormone auxin, target proteins can be rapidly degraded by the proteasome as previously described [55]. Genomic integration of auxin-specific TIR1 from Oryza sativa (OsTIR1) allowed us to degrade AID tagged fused proteins by auxin supplementation in yeast [51, 55]. To investigate the physiological impact of Lcb1, we used the AID system to deplete the essential protein in a dose-dependent way. As deletion results in nonviable yeast cells, we degraded Lcb1 endogenously tagged with AID, which was expressed in an OsTIR background strain [56]. For Lcb1-AID degradation, cells in stationary phase were diluted in fresh medium supplemented with 1 mM auxin.

Fluorescence microscopy

For localization studies cells expressing GFP-fusions of Tsc3p, ssSPTa and ssSPTb, were harvested in stationary phase and resuspended in phosphate buffered saline (PBS) after wash. For localization studies of Dga1-GFP in tsc3∆ cells, cells were harvested at indicated time points and fixed with 3.7% paraformaldehyde (PFA). After wash cells were resuspended in PBS. For analysis, 1 µl cell suspension was applied to microscopy slides, covered by 0.17 mm glass (Carl Zeiss, Jena, Germany). Images were captured using a confocal laser scanning microscope (LSM) 880 (Carl Zeiss, Jena, Germany) and processed with ImageJ 3.0.1 [57] or ZEN 2.3 (Carl Zeiss, Jena, Germany).

Assessment of LDs by flow cytometry

We used 96-well plates containing SC medium with 2% glucose (150 µl/well), which were inoculated with yeast strains of interest and precultured overnight (~ 12 h) by incubation at 30°C on a shaker. On the next day, the OD₆₀₀ was measured and cells were diluted in fresh medium to an OD₆₀₀ = 0.5. After further incubation for 24 h under the same conditions, the stationary phase of the cells was ensured by a static OD₆₀₀ value. Once stationary phase (T0) was confirmed, half volume of the cells was fixed 30 min with 3.7% PFA at room temperature and half volume was diluted and grown for 5 h (T5) in fresh SC containing 10 µg/ml cerulenin, which inhibits de novo fatty acid synthesis [58]. The incubation with cerulenin forces cells to use fatty acyls stored in LDs as new fatty acids cannot be produced. At the start of the experiment and after 5 h of incubation, the OD₆₀₀ was measured. Upon centrifugation at 4000 RPM (Swinging-bucket microplate rotor S6096 for Allegra X-30, Beckman Coulter, Brea, California/USA), the cells were collected, washed PBS, and fixed in 3.7% PFA. After fixation, the LDs of the cells were stained with 150 µl 1 µg/ml BODIPY 493/503 in PBS for 30 min in the dark. Subsequently, the cells were washed three times with PBS and resuspended in 120 µl appropriate buffer for flow cytometry measurements (BD FACSFlow sheat fluid BD Bioscience, Franklin Lakes, NJ/USA). The BODIPY 493/503 fluorescence signal of individual cells was analyzed by passing a detection laser using flow cytometry coupled to a 96-well plate high-throughput autosampler (BD FACSCalibur BD Bioscience).

Lipidome analysis by mass spectrometry

For lipidomics, 10 OD₆₀₀ units of cells were harvested from cultures of yeast grown in SC-medium containing 2% glucose for 24 h till reaching stationary phase and 5 h after dilution of stationary phase cells into fresh medium containing 10 µg/ml cerulenin [54, 59]. Cell pellets were washed once and lipid analysis were performed using a Triversa NanoMate ion source (Advion Bioscience Inc., Ithaca, NY/USA) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts/USA), as previously described [60, 61].

Total protein extraction

Total protein extraction from yeast to quantify cellular content of Lcb1-AID was conducted by alkaline lysis according to [54]. At indicated time points 2 OD₆₀₀ units of cultured cells were lysed in 0.25 M NaOH, 140 mM β-mercaptoethanol, 3 mM phenylmethylsulfonyl fluoride followed by 10 min incubation on ice. Proteins were precipitated by subsequent incubation with trichloroacetic acid (13% v/v). After centrifugation at 13000 RPM at 4°C total protein was washed with ice-cold acetone followed by resuspension in SDS sample buffer. Samples were analyzed by SDS-PAGE and western blotting. The following primary antibodies were used: Anti-HA (mouse monoclonal clone 16B12, 1:1000, BioLegend, San Diego, California/USA) and Anti-Pgk1 (mouse monoclonal clone 22C5D8, 1:5000, Thermo Fisher Scientific). IRDye 800CW Goat anti-Mouse IgG (Li-COR, 1:25000, Lincoln, Nebraska/USA) was used as secondary antibody

Statistical analysis

Data in figures are presented as means ± standard error of the mean (SEM) or standard deviation (SD). We performed one-way or two-way analysis of variance (ANOVA), and unpaired two-tailed t tests. Lipid data of Fig. S1 are from two independent experiments, analyzed in duplicates and are presented as mean ± SD. GraphPad Prism 8.2.1 was used to plot the graphs when comparing datasets (GraphPad Software Inc, San Diego, California/USA).

Statement specifying the appropriate permissions and/or licences for collection of plant or seed specimens

No plant or seeds were used in this study.

Data Availability Statement

M.H. and D.F.M. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors gave final approval of this version to be published.

The data that support the findings of this study are available from M.H. and D.F.M. but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Prof. Dr. Michael Roden, Scientific Executive Director of the Institute for Clinical Diabetology, German Diabetes Center.

Acknowledgments

We also thank Florian Fröhlich for donation of strain SEY6210 pRS305_OsTIR_3xFLAG and plasmid pBW2744- pHyg AID 71-114-6HA. This research was supported by the German Research Foundation (DFG), grant MA 7557/2-1 and the Villum Foundation (VKR023439, C.S.E.) and the VILLUM Center for Bioanalytical Sciences (VKR023179, C.S.E.).

Author contributions statement

M.H. wrote the article and researched the data. M.H. and D.F.M. designed and outlined experiments on Tsc3p characterization and researched the data. M.H. performed all laboratory analyses at the German Diabetes Center (DDZ), excluding the lipidomic analysis via mass spectrometry. Lipidomics analyses were conducted by F.M.M and C.S.E.

L.M., C.S.E, M.B., D.F.M., M.R., contributed to the discussion and reviewed the edited article.

Additional information

The authors declare no conflict of interest. All contributions to the manuscript by D.F.M. have been performed when affiliated to the German Diabetes Center, prior to current employment at Boehringer Ingelheim.

Farese, R.V., Jr. and T.C. Walther, Lipid droplets finally get a little R-E-S-P-E-C-T. Cell, 2009. 139(5): p. 855-60.
Fujimoto, T. and R.G. Parton, Not just fat: the structure and function of the lipid droplet. Cold Spring Harb Perspect Biol, 2011. 3(3).
Martin, S. and R.G. Parton, Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol, 2006. 7(5): p. 373-8.
Murphy, D.J., The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res, 2001. 40(5): p. 325-438.
Wilfling, F., et al., Lipid droplet biogenesis. Curr Opin Cell Biol, 2014. 29: p. 39-45.
Zhang, C. and P. Liu, The lipid droplet: A conserved cellular organelle. Protein Cell, 2017. 8(11): p. 796-800.
Greenberg, A.S., et al., The role of lipid droplets in metabolic disease in rodents and humans. J Clin Invest, 2011. 121(6): p. 2102-10.
Krahmer, N., R.V. Farese, Jr., and T.C. Walther, Balancing the fat: lipid droplets and human disease. EMBO Mol Med, 2013. 5(7): p. 973-83.
Apostolopoulou, M., et al., Specific Hepatic Sphingolipids Relate to Insulin Resistance, Oxidative Stress, and Inflammation in Nonalcoholic Steatohepatitis. Diabetes Care, 2018. 41(6): p. 1235-1243.
Lyu, K., et al., Short-term overnutrition induces white adipose tissue insulin resistance through sn-1,2-diacylglycerol/PKCepsilon/insulin receptor Thr1160 phosphorylation. JCI Insight, 2021. 6(4).
Petersen, M.C., et al., Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance. J Clin Invest, 2016. 126(11): p. 4361-4371.
Szendroedi, J., et al., Role of diacylglycerol activation of PKCtheta in lipid-induced muscle insulin resistance in humans. Proc Natl Acad Sci U S A, 2014. 111(26): p. 9597-602.
Fei, W., et al., Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast. J Cell Biol, 2008. 180(3): p. 473-82.
Kurat, C.F., et al., Cdk1/Cdc28-dependent activation of the major triacylglycerol lipase Tgl4 in yeast links lipolysis to cell-cycle progression. Mol Cell, 2009. 33(1): p. 53-63.
Mohammad, K., et al., Yeast Cells Exposed to Exogenous Palmitoleic Acid Either Adapt to Stress and Survive or Commit to Regulated Liponecrosis and Die. Oxid Med Cell Longev, 2018. 2018: p. 3074769.
Rockenfeller, P., et al., Fatty acids trigger mitochondrion-dependent necrosis. Cell Cycle, 2010. 9(14): p. 2836-42.
Rockenfeller, P., et al., Diacylglycerol triggers Rim101 pathway-dependent necrosis in yeast: a model for lipotoxicity. Cell Death Differ, 2018. 25(4): p. 767-783.
Fakas, S., et al., Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast. J Biol Chem, 2011. 286(33): p. 29074-29085.
Garbarino, J. and S.L. Sturley, Saturated with fat: new perspectives on lipotoxicity. Curr Opin Clin Nutr Metab Care, 2009. 12(2): p. 110-6.
Oelkers, P., et al., The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem, 2002. 277(11): p. 8877-81.
Oelkers, P., et al., A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem, 2000. 275(21): p. 15609-12.
Sorger, D. and G. Daum, Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae. J Bacteriol, 2002. 184(2): p. 519-24.
Czabany, T., K. Athenstaedt, and G. Daum, Synthesis, storage and degradation of neutral lipids in yeast. Biochim Biophys Acta, 2007. 1771(3): p. 299-309.
Gross, D.A. and D.L. Silver, Cytosolic lipid droplets: from mechanisms of fat storage to disease. Crit Rev Biochem Mol Biol, 2014. 49(4): p. 304-26.
Jacquier, N., et al., Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae. J Cell Sci, 2011. 124(Pt 14): p. 2424-37.
Mishra, S., et al., Mature lipid droplets are accessible to ER luminal proteins. J Cell Sci, 2016. 129(20): p. 3803-3815.
Ohsaki, Y., M. Suzuki, and T. Fujimoto, Open questions in lipid droplet biology. Chem Biol, 2014. 21(1): p. 86-96.
Casanovas, A., et al., Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism. Chem Biol, 2015. 22(3): p. 412-25.
Chauhan, N., et al., Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression. Proc Natl Acad Sci U S A, 2015. 112(10): p. E1077-85.
Nickels, J.T. and J.R. Broach, A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae. Genes Dev, 1996. 10(4): p. 382-94.
Sia, R.A., H.A. Herald, and D.J. Lew, Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast. Mol Biol Cell, 1996. 7(11): p. 1657-66.
Athenstaedt, K. and G. Daum, YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J Biol Chem, 2003. 278(26): p. 23317-23.
Athenstaedt, K. and G. Daum, Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles. J Biol Chem, 2005. 280(45): p. 37301-9.
Klein, I., et al., Regulation of the yeast triacylglycerol lipases Tgl4p and Tgl5p by the presence/absence of nonpolar lipids. Mol Biol Cell, 2016. 27(13): p. 2014-24.
Kurat, C.F., et al., Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast. J Biol Chem, 2006. 281(1): p. 491-500.
Schmidt, C., et al., Regulation of the yeast triacylglycerol lipase TGl3p by formation of nonpolar lipids. J Biol Chem, 2013. 288(27): p. 19939-48.
Carman, G.M. and S.A. Henry, Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes. Prog Lipid Res, 1999. 38(5-6): p. 361-99.
Fakas, S., C. Konstantinou, and G.M. Carman, DGK1-encoded diacylglycerol kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in Saccharomyces cerevisiae. J Biol Chem, 2011. 286(2): p. 1464-74.
McMaster, C.R., From yeast to humans - roles of the Kennedy pathway for phosphatidylcholine synthesis. FEBS Lett, 2018. 592(8): p. 1256-1272.
Brady, R.N., S.J. Di Mari, and E.E. Snell, Biosynthesis of sphingolipid bases. 3. Isolation and characterization of ketonic intermediates in the synthesis of sphingosine and dihydrosphingosine by cell-free extracts of Hansenula ciferri. J Biol Chem, 1969. 244(2): p. 491-6.
Rajakumari, S., K. Grillitsch, and G. Daum, Synthesis and turnover of non-polar lipids in yeast. Prog Lipid Res, 2008. 47(3): p. 157-71.
Cowart, L.A. and L.M. Obeid, Yeast sphingolipids: recent developments in understanding biosynthesis, regulation, and function. Biochim Biophys Acta, 2007. 1771(3): p. 421-31.
Hanada, K., Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim Biophys Acta, 2003. 1632(1-3): p. 16-30.
Gable, K., et al., Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity. J Biol Chem, 2000. 275(11): p. 7597-603.
Ren, J., et al., Tsc3 regulates SPT amino acid choice in Saccharomyces cerevisiae by promoting alanine in the sphingolipid pathway. J Lipid Res, 2018. 59(11): p. 2126-2139.
Han, G., et al., The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization, activity and localization. Biochim Biophys Acta Mol Cell Biol Lipids, 2019. 1864(3): p. 245-259.
Brice, S.E., C.W. Alford, and L.A. Cowart, Modulation of sphingolipid metabolism by the phosphatidylinositol-4-phosphate phosphatase Sac1p through regulation of phosphatidylinositol in Saccharomyces cerevisiae. J Biol Chem, 2009. 284(12): p. 7588-96.
Foti, M., A. Audhya, and S.D. Emr, Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology. Mol Biol Cell, 2001. 12(8): p. 2396-411.
Longtine, M.S., et al., Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 1998. 14(10): p. 953-61.
Markgraf, D.F., et al., An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER. Cell Rep, 2014. 6(1): p. 44-55.
Morawska, M. and H.D. Ulrich, An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast, 2013. 30(9): p. 341-51.
Serbyn, N., et al., The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast. Mol Cell, 2020. 77(5): p. 1066-1079 e9.
Robinson, J.S., et al., Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol, 1988. 8(11): p. 4936-48.
Ouahoud, S., et al., Lipid droplet consumption is functionally coupled to vacuole homeostasis independent of lipophagy. J Cell Sci, 2018. 131(11).
Nishimura, K., et al., An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods, 2009. 6(12): p. 917-22.
Giaever, G., et al., Functional profiling of the Saccharomyces cerevisiae genome. Nature, 2002. 418(6896): p. 387-91.
Schindelin, J., et al., Fiji: an open-source platform for biological-image analysis. Nat Methods, 2012. 9(7): p. 676-82.
Omura, S., The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis. Bacteriol Rev, 1976. 40(3): p. 681-97.
Ejsing, C.S., et al., Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc Natl Acad Sci U S A, 2009. 106(7): p. 2136-41.
Almeida, R., et al., Comprehensive lipidome analysis by shotgun lipidomics on a hybrid quadrupole-orbitrap-linear ion trap mass spectrometer. J Am Soc Mass Spectrom, 2015. 26(1): p. 133-48.
Martinez-Montanes, F., et al., Phosphoproteomic Analysis across the Yeast Life Cycle Reveals Control of Fatty Acyl Chain Length by Phosphorylation of the Fatty Acid Synthase Complex. Cell Rep, 2020. 32(6): p. 108024.
Kondo, N., et al., Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids. Nat Commun, 2014. 5: p. 5338.
Han, G., et al., Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities. Proc Natl Acad Sci U S A, 2009. 106(20): p. 8186-91.
Breslow, D.K., Sphingolipid homeostasis in the endoplasmic reticulum and beyond. Cold Spring Harb Perspect Biol, 2013. 5(4): p. a013326.
Davis, D.L., et al., The ORMDL/Orm-serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes. J Biol Chem, 2019. 294(13): p. 5146-5156.
Epstein, S., et al., Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells. J Lipid Res, 2012. 53(3): p. 412-420.
Epstein, S., et al., An essential function of sphingolipids in yeast cell division. Mol Microbiol, 2012. 84(6): p. 1018-32.
Matmati, N., et al., Hydroxyurea sensitivity reveals a role for ISC1 in the regulation of G2/M. J Biol Chem, 2009. 284(13): p. 8241-6.
Matmati, N., et al., Identification of C18:1-phytoceramide as the candidate lipid mediator for hydroxyurea resistance in yeast. J Biol Chem, 2013. 288(24): p. 17272-84.
Costanzo, M., et al., CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell, 2004. 117(7): p. 899-913.
Harmon, J.M., et al., Topological and functional characterization of the ssSPTs, small activating subunits of serine palmitoyltransferase. J Biol Chem, 2013. 288(14): p. 10144-10153.
Roden, M. and G.I. Shulman, The integrative biology of type 2 diabetes. Nature, 2019. 576(7785): p. 51-60.
Guillas, I., et al., C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p. EMBO J, 2001. 20(11): p. 2655-65.
Petschnigg, J., et al., Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast. J Biol Chem, 2009. 284(45): p. 30981-93.
Brachmann, C.B., et al., Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast, 1998. 14(2): p. 115-32.
Nguyen, T.T., et al., Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance. Traffic, 2012. 13(6): p. 880-90.
Prinz, W.A., et al., Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae. J Cell Biol, 2000. 150(3): p. 461-74.

No competing interests reported.

Supplementaryinformation.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

Yeast lipid droplet dynamics are coupled to sphingolipid biosynthesis via Tsc3p

Status:

Version 1

Abstract

Figures

Introduction

Results

Subcellular localization of the candidate protein Tsc3p

Discussion

Materials and Methods

Yeast strains and plasmids

Assessment of growth curve

Assessment of Lcb1 degradation by Auxin-inducible degron system

Fluorescence microscopy

Assessment of LDs by flow cytometry

Lipidome analysis by mass spectrometry

Total protein extraction

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1