Fig. 3: Regulation of SPT activity by Orm1 and ceramides.
a Ligand binding within the SPOT complex. The color code is the same as in Fig. 1. Ligands are represented as spheres with ergosterol in violet, 44:0;4 ceramide in dark blue, PLP in yellow, and GDN in semi-transparent gray. b Blocking of the substrate access tunnel by the putative Lcb2-gatekeeper residues Y110 and Y485 and ceramide 44:0;4, which is further stabilized by GDN. A docked 3-KS molecule (PDB: 7K0K, dotted black density) indicates the upper region of the substrate access tunnel. c Superposition of Orm1 and ORMDL3 (PDB: 7K0M) highlights divergence of M1-ORMDL3 towards the active site at Lcb2-K366 and Orm1-T39 towards Lcb1-E270. Diverging residues of ORMDL3 (V10-N11) and Orm1 (N74-P73) are marked with a triangle. Phosphorylation-sites are indicated with an orange triangle (residues mutated from serine to alanine). Other subunits were omitted for clarity. d Tetrad analysis of lcb2Δ cells (yellow), expressing Lcb2Y485S (green; upper panel) or Lcb2Y110S (blue) in the absence (middle panel) or presence of 20 µM PHS (lower panel). e Levels of 3-ketosphinganine (3-KS), long chain bases (LCBs), and ceramides in lcb2Δ cells expressing ALFA-Lcb2 or ALFA-Lcb2Y485S and WT cells expressing ALFA-Lcb2Y110S. Data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test (*p < 0.05, **p < 0.01, ***p < 0.001). Exact P-values are shown in Sup. Tab. 7. f Sequence alignments of the region surrounding the conserved Y residues in different species. g Serial dilutions of lcb2Δ orm1Δ cells expressing ALFA-Lcb2 and ALFA-Orm1, ALFA-Lcb2L69F and ALFA-Orm1M126F or ALFA-Lcb2L69F and ALFA-Orm1G122F on YPD plates (control) and YPD plates containing 2.5 µM myriocin. h Levels of 3-ketosphinganine (3-KS), long chain bases (LCBs) and ceramides in lcb2Δ orm1Δ cells expressing ALFA-Lcb2 and ALFA-Orm1 (left) or ALFA-Lcb2L69F and ALFA-Orm1M126F (middle) or ALFA-Lcb2L69F and ALFA-Orm1G122F (right). Data were analyzed by one-way ANOVA with Tukey’s multiple-comparison test (*p < 0.05, **p < 0.01, ***p < 0.001). Exact P-values for e and h are shown in Sup. Tab. 7. n = 4 biologically independent samples for e and h and data are presented as mean values ± SD. Source data are provided as a Source Data file for d, e, g, and h.
