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1 DOI: /ncb2480 a ~250 kda ~130 ~95 ~72 ~250 kda anti ypk1δ ypk2δ D239A S641A T659A tor2δ YPK2 ypk2δ CherryYPK1 unspecific crossreaction Cherry c b D239A S641A T659A YPK2 D239A S641A T659A tor2δ YPK2 ypk1δ ypk1 T662A WN Rap ~130 ~95 ~72 anti Cherry unspecific crossreaction psch9 T737 HASch9 d p MET3 ::GTR1 p MET3 ::gtr1 S20L met min e WN Myr min psch9 T737 HASch9 f fpk1 fpk2 Myr min g YPK2 D239A h FK506 Myr min Slm1GFP Figure S1 T662 phosphorylation is specifically mediated by TORC2 and requires Slm proteins. ab) The anti phospho T662 antiserum is specific. Total protein extracts from cells with the indicated genetic modifications were blotted, and membranes were probed with antisera recognizing phosphorylated on T662 or total. tor2 cells, in this experiment, are viable because they express the plasmidencoded hyperactive allele YPK2 D239A, S641A, T659A. cd) TORC1 and TORC2 specifically phosphorylate Sch9 T737 and T662, respectively. Rapamycin (Rap) treatment decreased Sch9 T737 phosphorylation while both Sch9 T737 and T662 phosphorylations were lost in cells treated with 10 µg/ml wortmannin (WN), which, at high concentrations, inhibits PI3kinase related kinases including Tor1/2 (c). Overexpression of the GTPase GTR1 from the methioninerepressed MET3 promoter increased Sch9 T737 phosphorylation, while overexpression of the GDPlocked gtr1 S20L mutant reduced Sch9 T737 phosphorylation (d). e) The increase of T662 phosphorylation during sphingolipid synthesis inhibition is not due to the inhibition of a phosphatase. Cells were treated with wortmannin (WN) for the indicated times to inhibit TORC2 activity. Prior treatment with myriocin (Myr) (right panels) did not retard the subsequent dephosphorylation of T662 demonstrating that inhibition of sphingolipid synthesis does not impair the activity of a T662 phosphatase. f) Activation of TORC2 during sphingolipid synthesis inhibition is independent of Fpk1/2 kinases. g) Expression of YPK2 D239A does not alter basal levels of T662 phosphorylation. h) Activation of TORC2 upon inhibition of sphingolipid synthesis is independent of calcineurin. Addition of the calcineurin inhibitor FK506 for the indicated times did not alter Myrinduced T662 phosphorylation. 1

2 a top sect. control Avo3GFP Lsp1RFPmars Myr AbA d Loewith Supplementary Figure 2 Slm1GFP Lsp1RFPmars control Myr AbA b c inlay inlay top sect. control GFP Lsp1RFPmars Slm1GFP Lsp1RFPmars Myr AbA Slm1GFP Pma1RFPmars W2469A W2473A tor2δ YPK2 D239A tor2 YPK2 D239A S641A T659A control top section mid section e ~170 kda ~100 ~70 ~55 ~40 M Input anti anti GFP anti HA Avo3HA Slm1GFP inlay Fluoresc. int. [a. u.] Slm1GFP Lsp1mars Distance [µm] Fluoresc. int. [a. u.] Slm1GFP Pma1mars Distance [µm] ~170 kda ~130 ~100 ~70 ~55 ~40 M GFPIP anti anti GFP anti HA Avo3HA Slm1GFP f YPD 1NMPP1 ypk2δ ypk2δ ypk1 L424G g ypk2δ 1NMPP min h Slm1GFP Lsp1RFPmars ypk2δ Slm1GFP Lsp1RFPmars ypk2δ ypk1 L424G NMPP1 inlay top sect. Figure S2 Slm1 dynamically relocalizes in and out of eisosomes. a) TORC2 plasma membrane localization, visualized by Avo3GFP (green) relative to Lsp1RFPmars (red) does not change during sphingolipid synthesis inhibition. Representative optical top sections are shown. Inlays depict magnified regions of interest (ROIs) of Avo3 (left), Lsp1 (middle) and overlay (right). Scale bar 5 µm. b) GFP (green) localization at the plasma membrane does not change during sphingolipid synthesis inhibition. Representative optical top sections are shown. Inlays depict magnified ROIs of (left), Lsp1 (middle) and overlay (right). Scale bar 5 µm. c) Slm1GFP (green) partially colocalizes with eisosomes (Lsp1RFPmars (red), left panel) but is excluded from the MCP (Pma1RFPmars (red), right panel). Representative optical mid and top sections are shown. Inlays depict magnified ROIs of Slm1 (left), Lsp1/Pma1 (middle) and overlay (right). Scale bar 5 µm. Plot profiles showing the relative fluorescent intensity of Slm1 GFP (green) and Lsp1/Pma1RFPmars (red) along the indicated lines of a representative optical top section. d) The release of Slm1GFP (green) from eisosomes (Lsp1RFPmars, red) observed upon inhibition of sphingolipid synthesis is independent of TORC2 activity. Representative optical top sections of strains with the indicated genetic background are shown. Inlays depict magnified ROIs of Slm1 (left), Lsp1 (middle) and overlay (right). Scale bar 5 µm. e) The physical interaction of Slm1 with TORC2 and increases following inhibition of sphingolipid synthesis. Full scans of western blots as shown in Fig. 2d are presented. f) The ATPanalogue 1NM PP1 specifically inhibits the analogue sensitive mutant ypk1 L424G. ypk2 ypk1 L424G cells are inviable when spotted on YPD plates containing 1NM PP1. g) Treatment with 1NMPP1 does not alter TORC2 activity in ypk2 control cells. h) ypk1 L424G inhibition triggers Slm1 exit from eisosomes. Slm1GFP (green) partially colocalizes with Lsp1RFPmars (red) in control and untreated cells, but relocalizes away from eisosomes following inhibition of ypk1 L424G by 1NMPP1. Representative optical top sections are shown. Inlays depict magnified ROIs of Slm1 (left), Lsp1 (middle) and overlay (right). Scale bar 5 µm. 2

3 TOR11 fpr1δ slm2δ SLM1FRB SUR72xFKBP Rap HATor2 MycAvo1 Avo3HA Figure S3 Anchoring Slm1 to eisosomes does not alter TORC2 stability. Rapamycin (Rap)mediated anchoring of Slm1 to eisosomes diminishes TORC2 activity but does not alter the protein levels of TORC2 components. 3

4 a 1 M sorbitol zymolyase zymolyase b top sect. control Slm1GFP Lsp1RFPmars Myr inlay Figure S4 Spheroplasting neither alters TORC2 activity nor Slm1 localization. a) TORC2 activity is unaffected during spheroplast preparation. T662 is similarly phosphorylated in cells grown in sorbitol medium (left lane), in cells after 15 min incubation in sorbitolcontaining buffer (middle lane), and in spheroplasts treated 15 min with zymolyase (right lane). b) Spheroplasting does not alter Slm1 (re)localization. Similarly to intact yeast cells, Slm1GFP (green) partially colocalizes with Lsp1RFPmars (red) in vehicle treated spheroplasts (control, left panels) but colocalization is lost after exposure to myriocin (5 µm, 60 minutes). Representative optical top sections are shown. Inlays depict magnified regions of interest (ROIs) of Slm1 (left), Lsp1 (middle) and overlay (right). Scale bar 5 µm. 4

5 a ORM1HA ypk1δ ORM1HA Myr λphos. ~40 kda ~35 kda * Orm1 PP HA Orm1HA b ORM1HA AbA Myr * Orm1 PP HA Orm1 P HA Orm1HA c ORM1HA ypk1 L424G ypk2δ ORM1HA ypk2δ Myr 1NMPP1 Orm1 PP HA * Orm1HA Figure S5 Orm1 phosphorylation increases upon inhibition of sphingolipid synthesis and requires kinase activity. a) is required for Orm1HA hyperphosphorylation upon inhibition of sphingolipid synthesis. Wild type or ypk1δ cells were treated with myriocin (Myr) as indicated. Cells extracts, treated or not with lamdaphosphatase (λphos.) were separated on phosphate affinity gels (see Methods) and Orm1HA phosphospecies were visualized by western blot. The asterisk indicates a nonspecific crossreaction of the antibody as observed in wild type cells not expressing the Orm1HA construct (first lane). b) Orm1HA phosphorylation increases when sphingolipid synthesis is pharmacologically inhibited with either aureobasidina (AbA) or myriocin (Myr). c) Myriocin (Myr)induced Orm1 hyperphosphorylation requires kinase activity as determined with cells expressing the analogue sensitive ypk1 L424G. 5

6 Supplementary Table Legends Supplementary Table 1: Yeast strains used in. Supplementary Table 2: Plasmids used in. Supplementary Movie Legends Supplementary Movie 1: Slm1 dynamically localizes to the plasma membrane and forms foci of different fluorescence intensity. Cells expressing Slm1GFP were imaged with TIRF microscopy. Images were acquired every 2 seconds over a period of 3.5 minutes. A representative optical top section is shown. Scale bar 1 µm. Supplementary Movie 2: Both pools of Slm1GFP are highly dynamic and proteins exchange between them. Cells expressing Slm1GFP were imaged with TIRF microscopy during FRAP experiments. Images were acquired every 2 seconds over a period of 3.5 minutes. A representative optical top section is shown. After 10 seconds, a single, immobile appearing Slm1GFP focus was bleached (white square) and its fluorescence recovery was followed over time. Scale bar 1 µm. Supplementary Movie 3: Slm1GFP leaves eisosomes upon mechanical membrane stretch. Spheroplasts expressing Slm1GFP and Lsp1RFPmars were imaged with spinning disc microscopy. A representative optical mid section is shown. The spheroplast was aspirated with a micropipette and plasma membrane suction was induced by applying a negative pressure. Images were acquired over a period of 25 seconds. Scale bar 1 µm. Supplementary Movie 4: Slm1GFP localization relative to eisosomes is altered after mechanical membrane stretch. Optical zstacks of spheroplasts expressing Slm1GFP (green, left) and Lsp1RFPmars (red, middle) were recorded before (upper panel) and after (lower panel) mechanical stretch experiments (as shown in Supplementary Movie 3). Representative 3D reconstructions are shown. 6

7 Supplementary Table 1: Yeast strains used in Strain Genotype Reference TB50a MATa trp1 his3 ura3 leu2 Wullschleger et al., 2005 YMP14 TB50a TRP1 ypk1 ::KAN YMP126 TB50a TRP1 ypk1 ::KAN prs416_ypk1 T662A YMP15 TB50a ypk2 ::HIS3 YMP1 TB50a tor2 ::KAN YEp352_YPK2 D239A, S641A, T659A HA YMP46 TB50a ypk1 ::KAN ypk2 ::HIS3 prs414 _CherryYPK1 YMP59 TB50a HIS3 TRP1 prs406_6hasch9 RL2145d TB50a fpk1 ::KAN fpk2 ::KAN YMP61 TB50a csg2 ::KAN prs406_6hasch9 YMP62 TB50a TRP1 LEU2 lcb3 ::HIS3 prs406_6hasch9 YMP63 TB50a sur1 ::KAN csg2 ::KAN prs406_6hasch9 YMP64 TB50a sur1 ::KAN prs406_6hasch9 YMP65 TB50a TRP1 HIS3 LEU2 ipt1 ::KAN prs406_6hasch9 YMP57 TB50a ypk1 ::KAN ypk2 ::HIS3 prs416_ypk1 YMP58 TB50a ypk1 ::KAN ypk2 ::HIS3 prs416_ypk1 L424G SEY6210 MATalpha leu23,112 ura352 his3 200 trp1 901 lys2801 suc2 9 Robinson et al., 1988 AAY1623 SEY6210 slm1 ::HIS3 slm2 ::HIS3 prs415_slm12 ts Audhya et al., 2004 TWY1758 AAY1623 YEp352_YPK2 D239A HA YMP79 SEY6210 YEp352_YPK2 D239A HA YMP80 SEY6210a slm1 ::HIS3 slm2 ::HIS3 YEp352_YPK2 D239A HA TWY138 MATa ura3 trp1 leu2 his3 ade2 can1100 Walther et al., 2006 TWY139 MATalpha ura3 trp1 leu2 his3 ade2 can1100 Walther et al., 2006 TWY1809 TWY138 Nat::p GALI::GFPSLM1 TWY776 TWY138 AVO3GFP::KAN LSP1RFPmars::Nat Berchtold and Walther, 2009 TWY775 TWY138 YPK1GFP::HIS3 LSP1RFPmars::Nat TWY798 TWY139 SLM1GFP::KAN LSP1RFPmars::Nat TWY1236 TWY138 SLM1GFP::HIS3 PMA1RFPmars::Nat TWY2506 TWY138 ypk1 ::Nat ypk2 ::Nat SLM1GFP::HIS3 LSP1RFPmars::Nat prs416_ypk1 L424G TWY2508 TWY138 ypk1 ::Nat ypk2 ::Nat SLM1GFP::HIS3 LSP1RFPmars::Nat prs416_ypk1 TWY2282 TB50a ADE2 AVO2TAP::TRP1 TOR2HA::KAN SLM1GFP::HIS LSP1RFPmars::Nat YEp352_YPK2 D239A HA TWY2286 TB50a ADE2 tor2 ::KAN SLM1GFP::HIS LSP1RFPmars::Nat YEp352_YPK2 D239A, S641A, T659A HA TWY2284 TB50a ade21 tor2 W2469A, W2473A HA::KAN SLM1GFP::HIS LSP1RFPmars::Nat YEp352_YPK2 D239A HA TWY1566 TWY138 AVO36HA::KAN TWY1627 TWY139 AVO36HA::KAN SLM1GFP::HIS3 LSP1RFPmars::Nat TWY2556 TWY138 AVO3GFP::KAN SLM12RFPmars::Nat TWY1883 TWY138 TOR11 fpr1 ::Nat TWY1991 TWY139 TOR11 fpr1 ::Nat slm2 ::hph SLM1FRBGFP::KAN TWY2533 TWY139 TOR11 fpr1 ::Nat slm2 ::hph SUR72FKBP12RFPmars::hph TWY2532 TWY138 TOR11 fpr1 ::Nat slm2 ::hph SLM1FRBGFP::KAN SUR72FKBP12RFPmars::hph TWY2500 TWY138 TOR11 fpr1 ::Nat slm2 ::hph SLM1FRBGFP::KAN SUR72FKBP12::HIS3 AVO36HA::KAN TWY2504 TWY2500 YEp352_YPK2 D239A HA YMP127 TWY2500 prs314_3hator2 YMP130 TWY2500 YEplac33_MycAVO1 TWY2563 TWY138 ORM16HA::HIS3 TWY2581 TWY138 ypk1 ::Nat ORM16HA::HIS3 TWY2580 TWY139 ypk1 ::Nat ypk2 ::Nat prs416_ypk1 ORM16HA::HIS3 TWY2569 TWY138 ypk1 ::Nat ypk2 ::Nat prs416_ypk1 L424G ORM16HA::HIS3 BY4741 MATa his31 leu2 met15 ura3 fps1::kanmx4 Brachman et al., 1998

8 Supplementary Table 2: Plasmids used in. Plasmid Reference YEp352_YPK2 D239A HA Kamada et al., 2006 YEp352_YPK2 D239A, S641A, T659A HA Kamada et al., 2006 pju996 prs406_6hasch9 pah126 prs416_ypk1 pah127 prs416_ypk1 L424G pmp26 prs414_cherryypk1 pas1 prs416_ypk1 T662A prs314_3hator2 Jiang and Broach, 1999 prl921 YEplac33_MycAVO1 Loewith et al., 2002 pju701 prs416_p MET3 ::GTR1 pju702 prs416_p MET3 ::gtr1 S20L