Pho85 and PI(4,5)P2 regulate different lipid metabolic pathways in response to cold

Abstract

Lipid homeostasis allows cells to adjust membrane biophysical properties in response to changes in environmental conditions. In the yeast Saccharomyces cerevisiae, a downward shift in temperature from an optimal reduces membrane fluidity, which triggers a lipid remodeling of the plasma membrane. How changes in membrane fluidity are perceived, and how the abundance and composition of different lipid classes is properly balanced, remain largely unknown. Here, we show that the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], the most abundant plasma membrane phosphoinositide, drop rapidly in response to a downward shift in temperature. This change triggers a signaling cascade transmitted to cytosolic diphosphoinositol phosphate derivatives, among them 5-PP-IP₄ and 1-IP₇, that exert regulatory functions on genes involved in the inositol and phospholipids (PLs) metabolism, and inhibit the activity of the protein kinase Pho85. Consistent with this, cold exposure triggers a specific program of neutral lipids and PLs changes. Furthermore, we identified Pho85 as playing a key role in controlling the synthesis of long-chain bases (LCBs) via the Ypk1-Orm2 regulatory circuit. We conclude that Pho85 orchestrates a coordinated response of lipid metabolic pathways that ensure yeast thermal adaptation.

Keywords: 1-IP(7); Low temperature; Orm2; Phosphoinositide; Phospholipid; Saccharomyces cerevisiae; Sphingoid bases; Sphingolipid; TORC2-Pkh1-Ypk1 signaling module; Triacylglyceride.

Fig. 1.

Schematic representation of the PPIn, IPs and DPIPs synthesis and its interacton with the Pho80-Pho81-Pho85 complex. The metabolic steps and enzymes involved in the synthesis and degradation of PI(4,5)P₂ are shown (see [10,12,13,35,36,37] for representative reviews). The hydrolysis of PI(4,5)P₂ by Plc1 generates IP₃, which is sequentially phosphorylated to form in the last steps the DPIP 1-PP-IP5 (1-IP₇) through the action of the IP₆ kinase Vip1 [PP-IP5 kinase or IP₇ kinase in mammals]. The 1-IP₇ isomer acts as an inhibitor of the cyclin-regulated kinase complex Pho81-Pho80-Pho85 [37,38], which responds to phosphate availability [39], and regulates, among others, the abundance and activity of the phosphatidate phosphatase Pah1 [41], a key enzyme in the synthesis of NLs and PLs [42]. The black dot indicates a single phosphate group. The red dot represents a high-energy phosphate or pyrophosphate. For more details see text.

Fig. 2.

Plasma membrane PI(4,5)P₂ levels lower in the cold-shocked cells. A) Cells of the wild-type CEN.PK2–1C strain were grown to the mid-logarithmic phase in SCD medium at 30°C. An aliquot was withdrawn for the analysis and the rest of the culture was shifted to 15°C for 3 h. Samples were analyzed for PI(4,5)P₂ levels by GC/MS. The values represent the mean (± SD) of at least three independent experiments (*; p< 0.05). B) The plasmid pRS414–7×2-PHO5-GFP-hPLC (see Table S3) which encodes a GFP-tagged PH domain was used to visualize the cellular location of yeast PI(4,5)P₂. Cells of the CEN.PK2–1C wild-type (wt) and the corresponding inp51 mutant strain were grown to the mid-logarithmic phase at 30°C. An aliquot was withdrawn for the analysis and the rest of the culture was shifted to 15°C for 3 h. Cells were concentrated and visualized as described in the Materials and Methods section. The cells photographed at 30°C and 15°C are shown as an example. Quantification of plasma membrane fluorescence was done with Image J and expressed as arbitrary units. Data are the mean (± SD) of at least three independent experiments in which 100 cells were processed per sample. The 15°C samples were significantly different compared with their respective control at 30°C (*; p< 0.05). The differences between the wt and the inp51 samples at the same temperature, 30°C or 15°C, were also statistically significant (#; p< 0.05). C) The GFP-PH levels from the protein extracts of the mentioned strains were analyzed by Western blot. Protein extracts were centrifuged at 17,900 × g for 10 min, and the corresponding pellet (P, membrane-enriched fraction) and supernatant (S, soluble fraction) fractions were separated by SDS-PAGE and immunoblotted with anti-GFP antibody. The cells from 30°C-grown cells (S0, P0) or transferred (3 h) to 15°C (S3, P3) were compared. The amount of G6Pdh and Pma1 were tested as loading control of cytosolic and membrane-associated proteins. Relative quantification of GFP signals in the inp51 mutant was done with Image J and is expressed as arbitrary units. The intensity of wild-type signals was under the detection limit. For more details see the Materials and Methods section. A representative experiment is shown.

Fig. 3.

DPIPs and Pho85 exert regulatory functions in response to cold. A) Cells of the CEN.PK2–1C wild-type (wt) strain and its corresponding plc1 mutant were grown as described above (Fig. 2A), and the expression of INO1 was analyzed by using an INO1-lacZ reporter as indicated in the Materials and methods section. Data represent the mean value (± SD) of three independent experiments (**; p< 0.01). B) The 1-IP₇ content in the cells of the wild-type and vip1 mutant strain was estimated by using a PHO89::lacZ reporter. Cells were grown at 30°C in SCD and then transferred to 15°C for 3 h. Data represent the mean value (± SD) of three independent experiments (**; p< 0.01). C) The activity of the repressible acid phosphatase Pho5 (Units/OD₆₀₀) was measured before and after transfer for 3 h of yeast cells from high-phosphate to phosphate-free medium. Cells of the wild-type, plc1, vip1 and pho85 mutants were analyzed. Data represent the mean value (± SD) of three independent experiments (**; p< 0.01).

Fig. 4.

Pah1 abundance and main inp51 phenotypes depend on Pho85. A) Cells of the CEN.PK2–1C wild-type (wt) strain and its corresponding mutants inp51, pho85 and inp51 pho85 were examined for growth in YPD lacking or containing 1.2 μM myriocin (YPD + Myr), 25 μM phytosphingosine (YPD + PhS), or exposed to 15°C (YPD 15°C). Toxicity of palmitoleic acid (C16:1) was tested on YPD-0.05% tergitol medium (YPD + Tergitol) supplemented with 750 μM of C16:1 (YPD + C16:1). Overnight YPD-grown cultures were adjusted to OD₆₀₀ ~ 0.5, diluted (1–10⁻³), spotted (3 μl) onto the mentioned media, and incubated at 30°C for 2–5 days. B) The protein extracts from Pah1-Myc tagged cells of the CEN.PK2–1C wild-type strain and its corresponding pho85 mutant were analyzed by Western blot. The 30°C-incubated cell cultures in High-phosphate medium (H Pi; OD₆₀₀ ~ 0.5) were transferred to phosphate starvation medium for 3 h (-Pi) or to 15°C for the same period (15°C). Antibodies against Myc and G6Pdh (loading control) were used. A representative experiment is shown.

Fig. 5.

Cold exposure affects lipid composition. A) Lipid profiles of wild-type cells in SCD were analyzed for NLs and PLs classes. 30°C-grown (OD₆₀₀ ~ 0.5) and 15°C-exposed (3 h) cells were analyzed by mass-spectrometry-based shotgun lipidomics. The amount of NLs, PLs, and their corresponding classes were normalized to the total lipid content and expressed as the mol%. B) The quantities of the PLs species containing the same number of double bonds or the same number of carbon atoms in the hydrocarbon moiety are summed and these values were normalized to the total amount of PLs and expressed as mol%. * (p< 0.05) and ** (p< 0.01) denote significant differences between 30°C and 15°C samples. Data represent the mean value (± SD) of three independent biological replicates.

Fig. 6.

Ypk1 and Orm2 are cold-regulated through control protein level. A) Schematic representation of the TORC2-Pkh1-Ypk1 signaling module. At the plasma membrane, the interaction of PI(4,5)P₂ and the PH-like domain-containing proteins Slm1/2 facilitates the targeting of Ypk1/2 to the plasma membrane and its later phosphorylation by the TORC2 complex and kinases Pkh1/2 [21,28], which also interact with PI(4,5)P₂. Activated Ypk1/2 stimulates the production of SLs by phosphorylating SPT inhibitor Orm2 [22,23] and ceramide synthase components Lag1 and Lac1 [24]. A black dot indicates a phosphate group. For more details, see the text. B) The protein crude extracts from the Ypk1-HA tagged cells of the CEN.PK2–1C wild-type strain and pho85 mutant were analyzed by Phos-tag affinity SDS-PAGE (upper panel) or regular SDS-PAGE (lower panel). Cells grown at 30°C in SCD medium, transferred to 15°C for 3 h (15°C) or exposed to 2 μM myriocin for 30 min (Myr) were examined. Protein extracts from 30°C-grown cells of the fpk1 fpk2 double mutant (fpk1/2) were used as controls. Images show only a part of the gels where Ypk1-HA isoforms were visualized with an anti-HA antibody. The slower migrating bands, which most likely correspond to the phosphorylated isoforms by Fpk1/2 (P-Ypk1-HA), are indicated. The band indicated by the arrow might be unspecific/phosphorylated and unphosphorylated Ypk1 isoforms (Ypk1-HA). The G6Pdh level was used as a loading control for crude extracts. A representative experiment is shown. C) Protein extracts from the Orm2-HA tagged cells of the wild-type and pho85 mutant strain were obtained by NaOH-treatment and analyzed by regular SDS-PAGE. Cultures at 30°C, transferred to 15°C for 3 h (15°C) or treated with 2 μM myriocin for the same time (Myr) were tested. The myriocin treatment stimulates the phosphorylation of Orm2 (P-Orm2-HA) by Ypk1 [31]. Membranes exposed for 30 and 60 s are shown. The G6Pdh level was used as a loading control for crude extracts. A representative experiment is shown. D) The ORM2 mRNA levels in cells of the CEN.PK2–1C wild-type (wt) strain and its corresponding pho85 mutant were analyzed by qPCR. Expression differences between wt and pho85 samples at 30 and 15°C (left panel) or between control (30°C) and cold-treated (15°C, 3 h) samples for the wt and pho85 strain (right panel) are shown as fold-change. Data represent the mean (± SD) of at least three independent experiments. Statistically significant (p< 0.05) differences are denoted (*, #).

Fig. 7.

A downward shift in temperature downregulates the synthesis of LCBs and stimulates its phosphorylation. A) The cells of the CEN.PK2–1C wild-type and corresponding pho85 mutant strain were grown in SCD medium at 30°C until the mid-log phase (OD₆₀₀ ~ 0.5) and were then transferred to 15°C for 3 h. Lipids were extracted, and the LCBs level was analyzed by mass spectrometry as described in the Materials and methods section. Data are the mean (± SD) of two independent biological replicates. Statistically significant differences (p< 0.05) between 30°C- and 15°C-samples (*), and between the wt and pho85 strains at the same temperature (#) are indicated. B) Schematic representation showing the initial steps of the SLs biosynthesis from the rate-limiting step catalyzed by the serine palmitoyltransferase (SPT) complex to ceramide synthase, and the LCBs-to-glycerophospholipid metabolic pathway. Orm2 acts as an inhibitor of SPT. LCBs kinases (Lcb4, Lcb5) use DhS and PhS, the precursors of DhC and PhC, to form their corresponding phosphorylated forms DhS-1P and PhS-1P. Details about each step, and the enzymes, effectors and regulators involved, can be found in the text and recent reviews [,–92,97]. C) Cells of the indicated strains were grown as indicated above and analyzed for LCBPs content. Statistically significant differences (p< 0.05) between 30°C- and 15°C-samples (*), and between the wt and pho85 strains at the same temperature (#) are indicated. D) The mRNA levels of the indicated genes in cells of the CEN.PK2–1C wild-type (wt) strain and its corresponding pho85 mutant were analyzed by qPCR. The expression changes in response to a cold-transfer from 30 to 15°C for 3 h are shown as fold-change for each strain. Data represent the mean (± SD) of at least three independent experiments. Statistically significant differences between wt and pho85 samples are denoted (#; p< 0.05).