Tsc3 regulates SPT amino acid choice in Saccharomyces cerevisiae by promoting alanine in the sphingolipid pathway

Abstract

The generation of most sphingolipids (SPLs) starts with condensation between serine and an activated long-chain fatty acid catalyzed by serine palmitoyltransferase (SPT). SPT can also use other amino acids to generate small quantities of noncanonical SPLs. The balance between serine-derived and noncanonical SPLs is pivotal; for example, hereditary sensory and autonomic neuropathy type I results from SPT mutations that cause an abnormal accumulation of alanine-derived SPLs. The regulatory mechanism for SPT amino acid selectivity and physiological functions of noncanonical SPLs are unknown. We investigated SPT selection of amino acid substrates by measuring condensation products of serine and alanine in yeast cultures and SPT use of serine and alanine in a TSC3 knockout model. We identified the Tsc3 subunit of SPT as a regulator of amino acid substrate selectivity by demonstrating its primary function in promoting alanine utilization by SPT and confirmed its requirement for the inhibitory effect of alanine on SPT utilization of serine. Moreover, we observed downstream metabolic consequences to Tsc3 loss: serine influx into the SPL biosynthesis pathway increased through Ypk1-depenedent activation of SPT and ceramide synthases. This Ypk1-dependent activation of serine influx after Tsc3 knockout suggests a potential function for deoxy-sphingoid bases in modulating Ypk1 signaling.

Keywords: HSAN1; Ypk1; ceramides; deoxysphingolipids; fatty acid; lipid signaling; lipidomics; mass spectrometry; serine palmitoyltransferase; sphingolipids; substrate selectivity; transferase.

Fig. 1.

Detection of d3KDS, dDHS, and dPHS using HPLC-ESI-MS/MS. HPLC separation of 0.625 pmol d3KDS (A), dDHS (B), and dPHS (C) followed by positive-ion-mode ESI-MS/MS. Ion intensities, RT, and parent/product ion pairs are shown in the legends to the right of the SRM peaks. Also shown are the chemical structures of each compound and their fragmentation scheme. Free LCBs from indicated yeast strains cultured in SD (D) or YPD (E) medium at 24°C to the log phase were analyzed. Amino acids were also extracted from these cultures, and the levels of alanine (F) and serine (G) are shown. Values shown on the bar graph are the means of three independent experiments, with the error bars representing standard deviations. RT, retention time; SRM, selected reaction monitoring.

Fig. 2.

Increased 3KDS and decreased d3KDS in tsc3Δ yeast. A: Measurement of LCB levels in WT, tsc3Δ, and orm1Δorm2Δ yeast strains. Yeast cells were cultured in SD medium to the late-log phase at 24°C. Cells were collected and subjected to lipid extraction followed by HPLC-ESI-MS/MS. B–F: Measurement of the levels of the indicated LCBs in WT, tsc3Δ, and tsc3Δ transformed with the WT TSC3 gene (tsc3Δ/TSC3). Cells were cultured in SD-leucine to the late-log phase before lipid extraction. The lipid measurement for each LCB species is represented as the mean from three independent cultures, with the standard deviation calculated using GraphPad Prism 5.

Fig. 3.

Increased l-serine (3,3)-D2 and decreased l-alanine (3,3,3)-D3 utilization by SPT in tsc3Δ yeast. A, B: Chemical structures of deuterated l-serine, l-serine (3,3)-D2, and deuterated l-alanine, l-alanine (3,3,3)-D3, and their SPT products D2-3KDS and D3-d3KDS. C, D: Increased l-serine (3,3)-D2 incorporation into D2-3KDS (C) and decreased l-alanine (3,3,3)-D3 incorporation into D3-d3KDS (D) in the tsc3Δ mutant. WT and tsc3Δ yeasts were cultured in SD medium to the log phase. l-Serine (3,3)-D2 or l-alanine (3,3,3)-D3 was added to the culture at 7.6 mM. Approximately 2–4 × 10⁸ of the cells were collected at the indicated time points for lipid extraction and LCB quantification by HPLC-ESI-MS/MS. Also shown are the incorporation of deuterated serine or alanine into orm1Δorm2Δ and orm1Δorm2Δtsc3Δ mutant yeast.

Fig. 4.

Tsc3 is required for alanine inhibition of serine incorporation into 3KDS by SPT. In vitro SPT activity assay with l-serine as a substrate using WT (A) and tsc3Δ microsomes (B) in the presence (dotted line) or absence of 7.6 mM alanine (solid line). SPT kinetics toward serine were measured by plotting the initial velocity of the reaction (the amount of D2-3KDS generated per microgram of microsomes per minute) against l-serine (3,3)-D2 concentrations used in the reaction ranging from 0 to 15.2 mM. Microsomes (200 µg) and 100 µM palmitoyl-CoA were used in all reactions. The data were fitted using the Michaelis-Menten equation and generated using GraphPad Prism 5. In the absence of alanine, the WT microsome SPT K_m value for serine was 2.0 ± 0.4 mM, and V_max was 160.0 ± 10.8 pmol/min/mg yeast microsome. In the presence of alanine, K_m for serine was 3.4 ± 0.6 mM, and V_max was 127.4 ± 9.4 pmol/min/mg yeast microsome. P values from the paired t-test comparing SPT velocity in the presence or absence of alanine are shown. *Significant difference between two groups of data.

Fig. 5.

Increased CerS activity in tsc3Δ. A: Accumulation of DHC (d18:0/26:0) and PHC (d18:0/26:0) in tsc3Δ. WT and tsc3Δ were cultured in SD medium to the late-log phase before lipid extraction and HPLC-ESI-MS/MS quantification of indicated ceramide species. The result shown is one representative from at least three independent experiments. B: The accumulation of DHC (d18:0/26:0) in tsc3Δ is reversed by introducing the WT TSC3 gene in the knockout strain. The indicated strains were cultured in SD-leucine medium to the late-log phase before lipid extraction and HPLC-ESI-MS/MS quantification of indicated ceramide species. Increased incorporation of C17-DHS into DHC (d17:0/26:0) (C) and PHC (d17:0/26:0) (E). C17-DHS was converted to C17-PHS at similar rate between WT and tsc3Δ (D). WT and tsc3Δ cells were cultured in SD medium to the log phase before the addition of 10 µM C17-DHS. Cells were collected at the indicated time points for lipid analysis.

Fig. 6.

Ypk1 is required for the activation of CerS and SPT in tsc3Δ. A–D: The increased formation of C17-DHC and C17-PHC from C17-DHS labeling in tsc3Δ is Ypk1-depenedent. A, B: WT and indicated mutant yeast strains were cultured to the late-log phase in SD medium and labeled with C17-DHS for 30 min. Lipids were extracted and subjected to HPLC-ESI-MS/MS to detect C17-DHC (A) and C17-PHC (B). Their relative amount was normalized by an internal standard and the amount of cells used (OD) as shown on the y-axis. The result shown is one representative from at least three independent experiments. C, D: WT, tsc3Δ, and tsc3Δypk1Δ were cultured to the log phase and labeled with 10 µM C17-DHS for the indicated time. The relative amount of C17-DHC (C) or C17-PHS (D) generated was plotted against labeling time. E: WT, tsc3Δ, and tsc3Δypk1Δ were cultured to the log phase and labeled with 7.6 mM l-serine (3,3)-D2 for the indicated time. The amount of D2-3KDS generated was plotted against the time points.

Fig. 7.

Tsc3 regulates the balance between canonical and noncanonical SPLs by promoting l-alanine incorporation into the SPL de novo synthesis pathway. A: Both alanine and serine are utilized by the intact SPT complex to maintain a homeostasis between canonical and noncanonical SPLs. Serine influx into canonical SPLs is at a faster rate than alanine incorporation into noncanonical SPLs. B: Eliminating the Tsc3 subunit from the SPT complex changes SPL homeostasis, with the generation of canonical SPLs becoming more dominant. The increased serine influx in tsc3Δ is partially due to the lack of alanine inhibition. The serine influx is also further boosted with Ypk1-dependent SPT derepression through Om1/2 deactivation and Ypk1-depenent CerS activation. The production of complex SPLs is not shown in these schemes. Enzymes in the SPL pathway are shown in gray boxes. SPL intermediate metabolites are shown in ovals with a blue outline. Ypk1’s action on its substrates are indicated with a red line (inhibition) and green arrow (activation). Steps of Ypk1-depdent increases in serine influx into the SPL pathway are shown with bold green arrows.