Supplementary Table 1: Metabolite concentration

METABOLITE SYMBOL

IN MODEL

CONCENTRATION (*) COMMENTS REFERENCE

3-Keto-Dihydrosphingosine

(KDHS) X1 5e-3 mol% (Φ) Low level expected because it is difficult to measure

levels for this metabolite by thin layer chromatography 1, (2, p 30693)

0.01 mol%

Ten fold the DHS-P concentration. Cowart A., personal communication. Data obtained by the procedures for mass and species measurements in Jenkins GM, et al., 2002

3 Dihydrosphingosine

(DHS) X2

0.53 mol% Exponential growth phase (1 × 107 cells/ml) (4, Table II)

0.036 mol% 5 Dihydroceramide

(Dihydro-C) X30.16 mol% Lag phase 6

0.001 mol% (7, Fig 3C) Dihydrosphingosine -1P (DHS-P) X4

0.00278 mol% Single measure for both S-1-P species (8, Table 2)

0.05 mol%

Ten fold the PHS-P concentration. Cowart A., personal communication. Data obtained by the procedures for mass and species measurements in Jenkins GM, et al., 2002

3 Phytosphingosine

(PHS) X5

0.16 mol% Exponential growth phase (1 × 107 cells/ml) (4, Table II)

0.005 mol% (7, Fig 3C) Phytosphingosine-1P (PHS-P) X6

0.00278 mol% Single measure for both S-1-P species (8, Table 2)

0.052 mol% 5

0.086 mol% Lag phase 6 Phytoceramide (Phyto-C) X7

4.5 mol% 2.5 A600 that correspond to 2.5-5 × 107 cells (9, Fig 8B)

0.102 mol% Value from Wu et al. at 30°C and 2 × 107 cells/ml. Estimate 10% non-plasma membrane concentration from Patton et al.

(10, Fig 7A), 11 Inositol

Phosphorylceramide (IPC-g)

X88.4 mol% Sum of IPC/C and IPC/D at 24°C (9, Fig 8B)

CDP- Diacylglycerol (CDP-DAG)

X9 5.4 mol% Complete synthetic medium (12, Table III)

8.4 mol% Microsomes (13, Table 2) Phosphatidylserine

(PS)

X10 9.8 mol% Complete synthetic medium (12, Table III)

- 1 -

3 mol% Microsomes (13, Table 2)

3.3 mol% Harvested in late log phase (14, Table 3) Phosphatidic Acid (PA) X11

3.1 mol% Complete synthetic medium (12, Table III)

Palmitoyl-CoA (Pal-CoA) X12 0.01 µM Low level for free long chain acyl-CoA esters (15, p 100)

2600 µM 16 Serine

X13 2720 µM Rabbit liver (17, Table 1)

0.1 mol% DAG and phospholipid concentrations for late exponential phase (18, Fig 1) sn-1,2-

Diacylglycerol (DAG)

X140.47 mol% Rat kidney 19

16.7 mol% Microsomes (13, Table 2)

4.6 mol% Exponential growth phase (2 × 107 cells/ml) (10, Fig 8) Phosphatidylinositol (PI) X15

7.5 mol% Complete synthetic medium (12, Table III)

Inositol (I)

X16 24.1 µM Cytosolic concentration (12, Table V)

Cytidine diphosphate- Ethanolamine

(CDP-Eth)

X17

22 µM

Estimated using the KM of DG-Ethanolamine phosphotransferase for CDP-Eth

Mannosylinositol Phosphorylceramide

(MIPC-g) X18

0.14 mol%

Value from Wu et al. at 30°C and 2 × 107 cells/ml. Estimate 10% non-plasma membrane concentration from Patton et al.

(10, Fig 7A), 11

0.0085 mol%

Value from Wu et al. at 30°C and 2 × 107 cells/ml. Estimate 10% non-plasma membrane concentration from Patton et al.

(10, Fig 7A), 11 Mannosyldiinositol

Phosphorylceramide ( M(IP)2C-g )

X194.2 mol% At 24°C and not all species measured (9, Fig 8B)

Plasma Membrane Inositol

Phosphorylceramide (IPC-m)

X20

0.918 mol%

Value from Wu et al. at 30°C and 2 × 107 cells/ml. Estimate 90% plasma membrane concentration from Patton et al.

(10, Fig 7A), 11

Plasma Membrane Mannosylinositol

Phosphorylceramide (MIPC-m)

X21

1.26 mol%

Value from Wu et al. at 30°C and 2 × 107 cells/ml. Estimate 90% plasma membrane concentration from Patton et al.

(10, Fig 7A), 11

Plasma Membrane Mannosyldinositol

Phosphorylceramide (M(IP)2C-m)

X22

0.0765 mol%

Value from Wu et al. at 30°C and 2 × 107 cells/ml. Estimate 90% plasma membrane concentration from Patton et al.

(10, Fig 7A), 11

0.5 mol% (20, Fig 2B) Very Long Chain Fatty Acid (C26-CoA)

X237.73 mol% Concentration of long and very long species C22:0, C24:0

and, C26:0 (21, Table VI)

183 µM 21.2 % of the Acetyl-CoA concentration according to the rat liver in vivo relationship between acetyl-CoA (68.5 µM) and Malonyl-CoA (14.5 µM)

22Malonyl-CoA

(Mal-CoA)

X24

1740 µM 1:2 Acetyl-CoA: Malonyl-CoA in vitro relation 23

- 2 -

870 µM Table 2 reported 2.50 mM/gr dry weight. Converted to µM using RSd Ysx from Table 4 and “O” from Fig 2

(24, Table 2 & 4) (25, Fig 2)

300 µM Escherichia coli (26, Table 4) Acetyl-CoA (Ac-CoA)

X25

300 µM Dictyostelium discoideum (27, Table 6) 1100 µM (28, Table II) Adenosine-5’-

Triphosphate (ATP)

X28 850 µM Permeabilized yeast cells (29, Fig 4)

3-Phosphoserine (3-P-Serine)

X37446 µM

Rabbit Liver (30, Table 1)

1176 µM Exponential growth (31, Fig 2) Glucose-6-P (G6P)

X47 1000 µM Permeabilized cells (29, Fig 4) Palmitate X58 0.05 µM Estimate

CoA X61 5 µM Rat liver physiological level 32

Acetate X62 0.01 µM

Calculated based on a low external acetate concentration of 1µM under aerobic exponential growth conditions: acetate pK of 4,75, internal pH of 6.75 and external pH of 4

(33, Fig 6 & eq 1)

(Φ) Parameter values in bold are used in the model.

- 3 -

Supplementary Table 2: Specific activities and kinetic parameters

ENZYME / TRANSPORTER SYMBOL GENE SPECIFIC

ACTIVITY (†) KINETICS COMMENTS REFERENCE

2.66e-3 U/mg (Φ) Microsomes 30,000 × g (13, Table 3)

8e-4 U/mg Microsomes (34, Table I) Phosphatidylinositol

Synthase (PI Synthase)

X26 PIS1

KM = 66 µM CDP-DAG KM = 210 µM Inositol

(35, Table VI)

2.62 e-4 U/mg (36, Table 5) 3-Ketodihydrosphingosine

Reductase (KDHS reductase)

X27

TSC10, YBR26

5w KM = 15 µM 3-KDS-SPH Beef liver microsomes (37, p 367)

5.4 e-6 U/mg (38, Table II)

KM = 0.036 mol% Dihydroceramide

Estimation value using dihydroceramide concentration

Dihydroceramide

Alkaline Ceramidase (Dihydro-CDase)

X29 YDC1

1.29e-4 U/mg KM = 3.84 mol% Dihydro-C Vmax = 1.2 U/mg Dihydro-C Rat brain (39, Table II &

p 27954)

0.0508 U/mg Mid-logarithmic phase (40, Table 1)

3.38 e-3 U/mg Using oleate as the fatty acid substrate (41, Table III)

Palmitoyl Transport & Palmitoyl-CoA

Synthase (Transp./ Palmitoyl CoA

Synthase)

X30

FAT1, FAA1, FAA4

KM = 20 µM Palmitoyl-CoA Vmax = 1.4 e-4 U/mg (42, Table I)

1.3 e-3 U/mg Specific activity of the same magnitude order as SHMT specific activity

(43, Table 2)

0.12 U/mg (44, Fig 3C)

0.78 U/mg KM = 20 µM 3-Phosphoserine Human recombinant enzyme (45, Table I)

Phosphoserine-Phosphatase

(P-Serine-PPase) X31 SER2

KM = 89 µM 3-Phosphoserine Rabbit liver (30, p 17)

4.5e-3 U/mg Cytosolic reversible enzyme (46, Table 2)

8.38e-3 U/mg Cytoplasm (47, Table II)

KM = 650 µM L-Serine Cytosolic (48, p 11)

Serine Hydroxymethyl Transferase

(SHMT) X32 SHM2

KM = 700 µM Serine (49, p 332)

- 4 -

3.3e-4 U/mg KM = 1.35 mol% Ceramide KM = 5 mol% PI

Microsomes. PI activates IPC synthase in a cooperative manner with a Hill constant of 3

(50, Table I)

2.1e-4 U/mg Microsomes in mid-exponential growth phase (51, Table I)

Ki = 2.89 mol% DHS Ki= 1.96 mol% PHS

Calculated based on IC50 data from Wu et al. and the MWC Dimeric model, interaction with effectors from Hayashi and Sakamoto

(10, Fig 10), 51, 52

Inositol Phosphorylceramide

Synthase (IPC Synthase)

X33 AUR1

KM = 0.5 mM PI Vmax = 6.6e-6 U/mg IPC-II and IPC-III (54, Fig 4)

1.65e-5 U/mg (10, p 13173)

K0.5 = 0.27 mol% DHS K0.5 = 0.2 mol% PHS Vmax = 135e-3 U/mg DHS Vmax = 105e-3 U/mg PHS

(55, p 2659)

Ceramide Synthase (Cer Synthase) X34

LAC1 LAG1

KM = 144 µM for DHS KM = 298 µM Behenoyl-CoA (C22:0)

Bovine liver mitochondria. Relation between the two KM is ~ 2 and is used for the estimation of the KM for C26-CoA using the KM DHS of Guillas et al.

(56, Table 2)

1.65e-4 U/mg

It was assumed that the activity of MIPC synthase is about 1/2 the IPC synthase activity

(54, Fig 4) Mannosyl Inositol Phosphoceramide

Synthase (MIPC Synthase)

X35SUR1 CSG1

KM = 0.102 mol% IPC Estimation of KM using IPC concentration

4e-6 U/mg

KM = 0.38 mol% DHS KM = 1.2 mol% PHS Vmax = 4.25e-6 U/mg DHS Vmax = 6e-6 U/mg PHS

(8, Fig 2 & 5)

Sphingoid Base Kinase X36LCB 4/5

0.0045 U/mg KM = 1.23 mol% (25 µM) ATP KM = 7.7 µM DHS KM = 10 µM PHS

Pellet results for activity. KM converted to mol% with the Lanterman and Saba relationship: 3.5 mol% = 1.8 mM

(57, Table II & Fig 2)

3.32 e-3 U/mg Microsomes (13, Table 3)

4.2e-4 U/mg (58, Table 3)

KM = 830 µM Serine KM = 83 µM CDP-DAG Ki = 65 µM I

(12, Table VI & p 18084)

Ki = 1.42 mol% DHS Ki = 3.55 mol% PHS

Calculated based on IC50 data from Wu et al. and the MWC Dimeric model, interaction with effectors from Hayashi and Sakamoto

(10, Fig 11), 52, 53

Phosphatidylserine Synthase

(PS Synthase) X38 CHO1

Ki= 7 mol% DAG Ka = 0.033 mol% PA Ka= 3.2 mol% PI

Kinetic order for PA and PI obtained from log-log plot from Fig. 3A (see Alvarez-Vasquez, et al. 2004)

53, (59, Table I and Fig 3A)

- 5 -

2.4e-3 U/mg (60, Table I)

3 U/mg KM = 50 µM Phosphatidic acid Vmax = 30 U/mg Late exponential phase (61, Table I)

KM = 2.2 mol% PA aKi = 0.4 mol% PHS aKi = 0.2 mol% DHS aKi= 1.5 mol% Sphingosine

(4, Table I) Phosphatidate Phosphatase (PA-PPase)

X39DPP1 LPP1

A0.5 = 2.6 mol% CDP-DG A0.5 = 5.5 mol% CDP-DAG

Kinetic order for CDP-DAG and PI obtained from log-log plot in this supplementary Information section 2

(62, Fig 4A and Fig 4B)

CDP-Diacylglycerol Synthase

(CDP-DAG Synthase) X40 CDS1 6.1e-4 U/mg

KM = 500 µM PA KM = 1 mM CTP Vmax = 4.7 U/mg

(63, Table 1)

8e-4 U/mg (64, Fig 3A) Sphingoid-1-phosphate

Phosphatase (SB-PPase)

X41LCB3 YSR2

KM = 8e-4 mol% (16 µM) DHS-P and PHS-P Vmax= 2.77e-5 µM

Rat liver microsomes. KM and Vmax converted to mol% with the Lanterman and Saba relationship: 3.5 mol% = 1.8 mM

8, (65, pag 382 and Fig 10)

DG-Choline Phosphotransferase

(ChoPT) X42 CPT1 6.6e-4 U/mg KM = 8 mol% Dioleoylglycerol (66, Table I and

Fig 3)

GPI Remodelase (Remodeling) X43 1e-4 U/mg KM = 0.052 mol%

Estimation of KM using phytoceramide concentration. Activity with order magnitude similar to IPC synthase

Phosphoinositol Kinase (PI Kinase) X44 VPS34 1.72 e-3 U/mg

KM = 70 µM PI KM = 300 µM MgATP Vmax = 4.75 U/mg

Microsomes (67, Table I and p: 18902)

DG-Ethanolamine Phosphotransferase

(EthPT)

X45 EPT1 1 e-3 U/mg

KM = 22 µM CDP-Eth KM = 3.3 mol% Dioleoylglycerol

(66, Table 1 and Fig 2)

8.33 e-4 U/mg

KM = 1180 µM Glucose-6-P KM = 8 µM NAD Vmax = 167 nmol/h

(68, Table I and p 7082) Inositol-1-P Synthase

(I-1-P Synth.) X46 INO1

3 e-4 U/mg Two hr after applied inositol to the medium (69, Fig 8)

Acyl-CoA-Binding Protein (ACBP)

X48 ACB1 20 µM Within the range 10 – 50 µM (70, Fig 2)

5.8 e-3 U/mg Microsomes 30,000 × g (71, Table 1)

1.04 e-3 U/mg Late logarithmic phase (72, Fig 2)

KM = 42 µM Palmitoyl-CoA Vmax =1. 53e-3 U/mg (73, Fig, 2B)

Activator effect of PS calculated in Alvarez-Vasquez, et al. , based on data of Mishra and Kamisaka

(53, p 283) (74, Fig 5)

Glycerol-3-Phosphate Acyltransferase

(G3P Acyltranferase) X49

GAT1, GAT2 ,SCT1

KM = 120 µM Glycerol-P KM = 4.6 µM Palmitoyl-CoA Escherichia coli (75, p 5)

- 6 -

3.67e-5 U/mg Average from different organisms (76, Table II)

Sphingosine-Phosphate Lyase

(Lyase) X50 DPL1

KM = 0.024 mol% (12.5 µM) DHS-P and PHS-P

Rat liver. Converted to mol% according to Lanterman and Saba: 3.5 mol% = 1.8 mM

8, (77, p 247)

Inositol Phosphosphingolipid

Phospholipase C (IPCase)

X51 X64

ISC1 1.5e-4 U/mg KM = 3.57 mol% IPC KM = 1.85 mol% MIPC KM = 1.92 mol% M(IP)2C

X51 correspond to the IPCase with Phytoceramide as product and X64 to the Dihydro-C as product

(78, Table I and II)

8.9 e-3 U/mg KM = 8 µM Mal-CoA KM = 28 µM Ac-CoA

(79, Table I and p 22)

8.4e-3 U/mg (80, Table 2) 3.124 e-3 U/mg (81, Table I)

0.065 U/mg (82, Table 1)

Fatty Acid Synthetase (FAS) X52

FAS1, FAS2, FAS3

KM = 6 -19 µM Ac-CoA Cryptococcus neoformans (83, Table 2) 1.98e-5 U/mg (38, Table II)

KM = 0.052 mol% (*)

Phytoceramide Assumed similar to phytoceramide concentration

Phytoceramide Alkaline

Ceramidase (Phyto-CDase)

X53 YPC1 KM = 1.29 mol% Ceramide Rat brain (39, Fig 7A)

1.7e-4 U/mg Microsomes (84, Table II) 4-Hydroxylase (Hydroxylase)

(SYR2p – SUR2p)

X54SYR2 SUR2

KM = 0.01 mol% DHS KM = 0.036 mol% Dihydroceramide

Assumed similar to substrate concentration

8.25e-5 U/mg Activity of M(IP)2C synthase assumed to be about 1/4 the IPC synthase activity

(54, Fig 4) Mannosyldiinositol Phosphorylceramide

Synthase (M(IP)2C Synthase)

X55 IPT1 KM = 0.14 mol% MIPC Estimation of KM using

MIPC-g concentration

1.06e-5 U/mg PSD2p value for non-mitochondrial enzyme (85, Table I)

KM = 24 µM PS E. coli (86, p 3081 and Fig 3)

Phosphatidylserine Decarboxylase

(PS Decarboxylase) X56 PSD2

KM = 9.5 µM PS (87, p :6064)

1.06e -4 U/mg

KM = 4000 µM Serine KM = 15 µM Palmitoyl-CoA Vmax = 1.03 e-4 U/mg

KM for Palmitoyl-CoA required correction because of incorrect calculations in the insert of Fig 3A

(36, Table 3 and Fig 3A)

Serine Palmitoyltransferase

(SPT)

X57LCB1/2 SCS 1

6.31e -5 U/mg KM = 670 µM Serine Rat liver (88, Table I)

Very Long Chain Fatty Acid Synthase

(ELO1p) X59 ELO1 6e-4 U/mg KM = 130 µM Mal-CoA

KM = 130 µM Pal-CoA 89

- 7 -

0.022 U/mg KM = 16 µM Ac-CoA KM = 660 µM ATP

(90, p 7)

0.009 U/mg

Ki= 6.5e-3 µM Pal-CoA, C16:0

Ki= 4.8e-2 µM Arachidonoyl-CoA, C20:0 KM = 25 µM Ac-CoA KM = 15 µM ATP

Rat liver (91, Table I and II & p 12)

Ki = 5.5e-3 µM Pal-CoA and C26-CoA Liver (92, Table 2)

KM = 19 µM Ac-CoA Ki = 7.2 µM Palmityl-CoA Ki = 1.3 µM Oley-CoA

Rat liver (93, Table 4)

0.029 U/mg (94, Table II) 0.042 U/mg (95, Table 2) 0.0025 U/mg (82, Table 1)

Acetyl-Coenzyme A Carboxylase

(ACCp) X60 ACC1

KM = 260 µM Ac-CoA Candida lipolytica (96, p 42)

0.73 U/mg KM = 208 µM Acetate KM = 238 µM CoA

Activity for the microsomal fraction. Kinetic order for Pal-CoA inhibition obtained from log-log plot of Fig 1

(97, Table 2 and Fig 1)

0.22 U/mg KM = 1100 µM ATP KM = 600 µM Acetate Vmax = 1.2

(98, Table I)

0.66 U/mg KM = 35 µM CoA KM = 280 µM Acetate KM = 1200 µM ATP

(99, Table IV)

Acetyl-Coenzyme A Synthetase

(ACSp) X63

ACS1, ACS2

0.025 U/mg (100, Table II)

(Φ) Parameter in bold are used in the model. (*) mol% = concentration of sphingoid base or phosphatidate / concentration of total phospholipid. (†) U/mg = µmol/min/mg

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