Total Molar Balances

Supplementary information

The steady state equations used to solve the network shown in the Fig. 1

Total molar balances

FLO11t = FLO11 + FLO11_Flo8p_Ste12pTec1p + FLO11_Flo8p + FLO11_Ste12pTec1p +

Sfl1_FLO11

Flo8t = Flo8 + Flo8p + FLO11_Flo8p + Flo8p_E2 + Flo8_Tpk2 + FLO11_Ste12pTec1p_Flo8p

Sfl1t = Sfl1 + Sfl1p + Sfl1_FLO11 + Sfl1p_E4 + Sfl1_Tpk2

E2t = E2 + Flo8p_E2

E4t = E4 + Sfl1p_E4

E6t = E6 + Ste12pTec1p_E6

Adct = Adc + Adc_Ras2GTP + Adc_Gpa2GTP

Ste12Tec1_t = Ste12Tec1 + Ste12pTec1p + Ste12pTec1_Kss1p + Ste12Tec1_Tpk1 +

Ste12Tec1_Tpk2+ Ste12Tec1_Tpk3 + Ste12pTec1p_E6

+FLO11_Flo8p_Ste12pTec1p + FLO11_Ste12pTec1p

Pde1t = Pde1 + Pde1p_cAMP + Pde1p + Pde1p_E8 + Pde1_tpk1+ Pde1_tpk3

Pde2t = Pde2(1+cAMP/Km14)

R2C2t = R2C2 + R2(cAMP)4

R2C2t represents total PKA complex of Tpk1, Tpk2, Tpk3 with Bcy1(R)

Ct = 2 R2C2 + C + Pde1_tpk1 + Pde1_tpk3 ( where C = tpk1 + tpk2+ tpk3) cAMPt = cAMP + R2(cAMP)4 + Pde1p_cAMP + Pde2_cAMP

Equilibrium Relations

[FLO11_ Flo8p] [FLO11_ Ste12 pTec1p] Kd1  Kd 2  [Flo8p][FLO11] [Ste12 pTec1p][FLO11] [FLO11_ Ste12pTec1p _ Flo8p] [FLO11_ Sfl1] Kd3  Kd4  [Flo8p][Ste12 pTec1p][FLO11] [Sfl1][FLO11]

[Adc _ Gpa2GTP] [Adc _ Ras2GTP] Kd5  Kd6  [Gpa2GTP][Adc] [Ras2GTP][Adc]

4 [cAMP] [R2C2 ] Kd7  2 [R2 (cAMP)4 ][C]

Rate expression with pseudo-steady state representation of complexes for

modification-demodification cycles

Flo8 activation by Tpk2

k1 k 2 [Flo 8][ Tpk 2]= [ Flo 8 p ][ E 2] Km1 Km 2

Inhibitor inactivation by Tpk2

k3 k4 [Sfl1][Tpk2]  [Sfl1p][E4] Km3 Km4

Ste12Tec1 activation by Tpk1/2/3 and Kss1p

Tpk1 Tpk2 Tpk3 Kss1p  E6 k5[Ste12Tec1]     k6 [Ste12 pTec1p]  Km5 Km6 Km7 Km8  Km9 Pde1 activation by Tpk1 and Tpk2

 Tpk1 Tpk3  E8 k7 [Pde1]   k8[Pde1p]  Km10 Km11 Km12

cAMP rate balance

k10 k11 k9 [Adc _ a]  [Pde1p][cAMP]  [Pde2][cAMP] Km13 Km14 Activation of Kss1p through MAPK pathway ( Ras2GTP)

nH Kss1max Ras2GTP Kss1p  nH nH K 0.5  Ras2GTP The steady equations including the effect of Tpk1 and Tpk3 on Flo8 and Sfl1

Molar Balance

Flo8t = Flo8 + Flo8p + FLO11_Flo8p + Flo8p_E2 + Flo8_Tpk1 +Flo8_Tpk2 + Flo8_Tpk3+

FLO11_Ste12pTec1p_Flo8p

Sfl1t = Sfl1 + Sfl1p + Sfl1_FLO11 + Sfl1p_E4 + Sfl1_Tpk1+Sfl1_Tpk2 + Sfl1_Tpk3

Flo8 activation by Tpk1, Tpk2, Tpk3

Tpk1p  k12 Tpk2 p  k1 Tpk3p  k13 E2 [Flo8]    k2[Flo8p]  Km15 Km1 Km16  Km2 Inhibitor inactivation by Tpk1, Tpk2 Tpk3

Tpk1p  k14 Tpk2  k3 Tpk3 k15 E4 [Sfl1]    k4 [Sfl1p]  Km17 Km3 Km18  Km4

Component Concentrations

FLO11t = 0.1 nM (Malkey et al. 2003), Ste12Tec1_t = 200nM (Kofhal et al. 2004),

Flo8t = 150nM (Neelajan et al. 2007), Sfl1t = 150nM (Neelajan et al. 2007), Adct =

40nM (Bhalla et al. 2002), R = 1000nM (Bhalla et al. 2002), C = 1000nM (Bhalla et al.

2002), cAMPt = 7000nM (Bhalla et al. 2002), Pde1t =100 (Neelajan et al. 2007), Pde2t =

50 (Neelajan et al. 2007), Kss1max = 100 (Neelajan et al. 2007).

All the phosphatases concentrations are in the range as reported by Huang et al.

1996. They are further chosen on basis of two criteria: first the activation should not be restricted below 90% and second the cascades are saturated so that the phosphatase concentration is roughly 1/10 of the kinase concentration (Koshland et al. 1982).

E2t= 10 nM, E4t = 10 nM, E6t = 10 nM, E8t =10nM

Dissociation constants

Kd1 = Kd2 =10nM (assumed), Kd3 =100nM2 (assumed), Kd4 =10nM (assumed), Kd5 =

2 nM (Bhalla et al. 2002), Kd6 =10 nM (Shima et al. 1997), Kd7 = 2.634x106 nM2(Bhalla et al. 2002).

Rate constants (min-1) k1 = 150, k2 =150, k3 = 150, k4 = 150, k5 = 150, k6 =150, k7 =150, k8 = 150 (Huang et al 1996), k9 = 1040, k10 =1200, k11 = 600 (Balla et al. 2002) k12/Km15 = 0.05, k13/Km16 = 0.05 – Activation of Flo8p by Tpk1/3 k14/Km17 = 1x10-3, k15/Km18 = 1x10-3 – Inhibitor inactivation by Tpk1/3

Michaelis Menton constants (Huang et al. 1996)

Km1 = 100nM, Km2 =100nM, Km3 = 100nM, Km4 = 100nM, Km8 =100nM, Km9 =

100nM, Km10 = 100nM, Km11 = 100nM, Km12 =100nM, Km13=100nM,

Km14=100nM, Km5 =Km6 = Km7 = 100-1000nM

Supplementary references

Bhalla US. (2002) Use of Kinetikit and GENESIS for modeling signaling pathways. Methods Enzymol 345: 3–23.

Huang CYF and Ferrell Jr JE. (1996) Ultrasensitivity in the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 93: 10078-10083. Malkhey V, Bhat PJ and Venkatesh KV. (2003) Quantitative analysis of GAL genetic switch of Saccharomyces cerevisiae reveals that nucleocytoplasmic Shuttling of Gal80p results in a highly sensitive response to galactose. J Biol Chem 278: 48764–48769.

Kofahl B and Klipp E. (2004) Modelling the dynamics of the yeast pheromone Pathway. Yeast 21: 831–850.

Shima F, Yuriko YK , Yanagihara C, Tamada M ,Tomoyo O, Kariya K and Kataoka T. (1997) Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein. Mol Cell Biol 17:1057–1064.