Orm proteins control ceramide synthesis and endocytosis via LCB-mediated Ypk1 regulation

Abstract

Sphingolipids (SPLs) are major components of cell membranes with significant functions. Their production is a highly-regulated multi-step process with the formation of two major intermediates, long chain bases (LCBs) and ceramides. Homologous Orm proteins in both yeast and mammals negatively regulate LCB production by inhibiting serine palmitoyltransferase (SPT), the first enzyme in SPL de novo synthesis. Orm proteins are therefore regarded as major regulators of SPL production. Combining targeted lipidomic profiling with phenotypic analysis of yeast mutants with both ORM1 and ORM2 deleted (orm1/2Δ), we report here that Ypk1, an AGC family protein kinase, signaling is compromised in an LCB-dependent manner. In orm1/2Δ, phosphorylation of Ypk1 at its activation sites is reduced, and so is its in vivo activity shown by reduced phosphorylation of Ypk1 substrate, Lac1, the catalytic component of ceramide synthase (CerS). A corresponding defect in ceramide synthesis was detected, preventing the extra LCBs generated in orm1/2Δ from fully converting into downstream SPL products. The results suggest that Orm proteins play a complex role in regulating SPL production in yeast S. cerevisiae by exerting an extra and opposite effect on CerS. Functionally, we define endocytosis and an actin polarization defect of orm1/2Δ and demonstrate the roles of Ypk1 in mediating the effects of Orm proteins on endocytosis. Collectively, the results reveal a previously unrecognized role of yeast Orm proteins in controlling ceramide synthesis and their function in endocytosis through regulating Ypk1 signaling.

Keywords: LCBs; Orm1/2; SPT; Ypk1; cell signaling; endocytosis; enzymology/enzyme regulation; lipidomics; lipids; sphingolipids.

Fig. 1

Schematic diagram of sphingolipid metabolic pathway in yeast S. cerevisiae. The chemical structures of complex SPLs and the major intermediates, including LCBs, ceramides and LCB-1-Ps, are shown. Sphingosine backbone, acyl chain of ceramides, and a head group of complex SPLs are labeled in gray, orange, and red, respectively. P in purple circle marks the phosphate group. Also shown are their cellular distribution and relative abundance. Blue ovals indicate enzymes required for the synthesis of LCBs and ceramides, respectively.

Fig. 2

Deletion of ORM1/2 has different effect on cellular level of LCBs versus ceramides and complex SPLs. A: A simplified diagram of de novo synthesis pathway of sphingolipids in S. cerevisiae. The major groups of SPL species measured in this study are shown. SPT and CerS are highlighted. Deoxy-SPLs derived from alanine are in red. B–I: SPL profile of WT versus orm1/2Δ. Yeast cells were cultured in YPD medium to exponential growth phase, whereafter lipids were extracted and quantified via HPLC-ESI-MS/MS. J: Fold changes in indicated SPL species in orm1/2Δ versus WT. The data are shown as mean with SEM and were analyzed using an unpaired two-tailed t test in GraphPad Prism5. ∗P < 0.05. 3KDS, 3-ketodihydrosphingosine; C26-PHC, C26-phytoceramide; C26-PHC-OH, hydroxy C26-phytoceramide; C26-PHC-OH-IPC, inositol phosphoryl C26-PHC-OH; C26-PHC-OH-MIPC, mannose-inositol-phosphoryl C26-PHC-OH; C26-PHC-OH-M(IP)2C, mannose-(inositol-P)₂-C26-PHC-OH; DHC, dihydroceramide; DHS, dihydrosphingosine; DHS-1-P, dihydrophingosine-1-phosphate; IPC, inositol phosphorylceramide; MIPC, mannosylinositol phosphorylceramide; M(IP)2C, mannosyldiinositol phosphorylceramide; PHS, phytosphingosine; PHC, phytoceramide; PHS-1-P, phytosphingosine-1-phosphate.

Fig. 3

orm1/2Δ mutants are defective in ceramide production. A: Incorporation of L-Ser (3,3)-D2 into deuterated (D2-) LCBs and ceramides. B: The ratio between D2-C26-PHC and D2-PHS was plotted at each time point to reflect CerS activity. C: Conversion of C17-PHS into C17-PHS-1P and C26-PHC(d17). 7.6 mM D2-Ser (A) or 10 μM C17-PHS (C) were added to the yeast culture. Yeast cells were collected at indicated time points before being subjected to lipid extraction and quantification by HPLC-ESI-MS/MS. The data shown are representative results of three independent experiments. D, E: Deoxy-SPL profile of WT versus orm1/2Δ. Yeast cells were cultured in a synthetic defined growth medium to exponential growth phase before being subject to lipid extraction and quantification via HPLC-ESI-MS/MS. dSa, deoxy-sphinganine.

Fig. 4

Orm proteins regulate CerS activity through PHS-modulated Ypk1 activation. A: Lac1 phosphorylation is decreased in orm1/2Δ yeast and can be corrected by deleting TSC3. Whole-cell extracts of the indicated strains containing Flag-Lac1 were resolved by phos-tag SDS-PAGE and immunoblotted with an anti-Flag antibody. B: LCB accumulation in orm1/2Δ yeast is abolished by deleting TSC3. Lipids were extracted and PHS level was measured with HPLC-ESI/MS/MS. C: Exogenously added PHS reduces Lac1 phosphorylation. WT yeast containing Flag-Lac1 were cultured to exponential phase and treated with 10 μM PHS at the indicated time points. D: Ypk1 phosphorylation is decreased in orm1/2Δ and can be corrected by deleting TSC3. Whole-cell extracts of the indicated strains were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. E: Exogenously added PHS reduces Ypk1 phosphorylation. Indicated strains were cultured to exponential phase and treated with or without 10 μM PHS treatment for 5 min. F: Ypk2^D239A rescues the Lac1 phosphorylation defect in orm1/2Δ yeast. Whole-cell extracts of the indicated strains containing Flag-Lac1 were resolved by phos-tag SDS-PAGE and immunoblotted with an anti-Flag antibody. Band intensity measured using image J were used to calculate ratios between Lac1-P and Lac1 and are shown on the right. G: A diagram illustrating the relationships deduced from these results between Orm1/2/LCBs and Ypk1 on CerS. Western Blot shown here are representative of three biological replicates. ∗P < 0.05.

Fig. 5

Defining an endocytosis defect of orm1/2Δ. Defective endocytosis of Lucifer yellow (A), FM4-64 (B), and Ste2-GFP (C) in orm1/2Δ yeast. Indicated yeast strains stained with Lucifer yellow, FM4-64 or containing GFP-tagged Ste2 were observed under a fluorescence microscope. These images are representative of three independent biological replicates. The graph beneath each figure shows the percentage of cells with normal accumulation of the indicated endocytic markers. Three randomly selected fields were counted blindly for each strain. ∗P < 0.05.

Fig. 6

Actin polarization is defective in orm1/2Δ yeast. Indicated yeast were fixed and stained for actin with Rhodamine-phalloidin. Arrows of different forms point to cells at different stages of the cell cycle. Three fields of cells were randomly selected and visualized under both DIC and fluorescence optics. The graph on the right shows the percentage of cells with polarized actin patches.

Fig. 7

A model illustrating Orm1/2-LCB-Ypk1 signaling pathway. Orm proteins regulate LCB production to control Ypk1 and downstream SPL production and endocytosis. Phosphorylation of Orm1/2 by Ypk1 might serve as a feedback mechanism to reduce the activation signal.