Table 1. Yeast strains used in this study Strain

Genotype

Source

S18-1D

MATα his3 leu2 ura3 trpl ade8 tpk1w tpk2::HIS3

Ref.3

tpk3::TRP1 TP408a

S18-1D krh1::URA3

This study

TP626a,b

S18-1D krh1::URA3 krh2::KanMX4

This study

RS13-58A-1

MATa his3 leu2 ura3 trpl ade8 tpk1w tpk2::HIS3

Ref.3

tpk3::TRP1 bcy1::LEU2 TP415a

RS13-58A-1 krh1::URA3

This study

TP627a,b

RS13-58A-1 krh1::URA3 krh2::KanMX4

This study

BY4742

MATα leu2 lys2 ura3 his3

Ref.4

Y10351

MATα leu2 lys2 ura3 krh1::KanMX4

Ref.5

Y11668

MATα leu2 lys2 ura3 his3 krh2::KanMX4

Ref.5

Y00351

MATa leu2 met15 ura3 his3 krh1::KanMX4

Ref.5

TP261d

MATα krh1::KanMX4 krh2::KanMX4

This study

Y10152

MATα leu2 lys2 ura3 his3 gpa2::KanMX4

Ref.5

TP675c

TP261 gpa2::LEU2

This study

TP676k

TP261 GPA2(Q300L)::LEU2

This study

TP677k

BY4742 GPA2(Q300L)::LEU2

This study

TP380a,e,f

MATa/α cyr1::KanMX4/CYR1 pde2::URA3/PDE2

This study

krh1::LEU2/KRH1 krh2::HIS3/KRH2 TP390g

cyr1::KanMX4 pde2::URA3 krh1::LEU2 krh2::HIS3

This study

TP420e,f

MATa/α cyr1::KanMX4/CYR1 pde2::URA3/PDE2

This study

TP433h

MATα cyr1::KanMX4 pde2::URA3

This study

TP522a,e,f,i

MATa/α cyr1::KanMX4/CYR1 pde2::URA3/

This study

pde2::URA3 krh1::HIS3/KRH1 RAS2(G19V)::LEU2 TP519j

cyr1::KanMX4 pde2::URA3 RAS2(G19V)::LEU2

This study

TP467e,f,k

MATa/α cyr1::KanMX4/CYR1 pde2::URA3/PDE2

This study

GPA2(Q300L)::LEU2 TP506l

cyr1::KanMX4 pde2::URA3 GPA2(Q300L)::LEU2

This study

TP538a,m

MATa/α tpk1::KanMX4/tpk1::KanMX4

This study

tpk2::KanMX4/tpk2::KanMX4 tpk3::LEU2/TPK3 yak1::URA3/YAK1 TP543n

MATα tpk1::KanMX4 tpk2::KanMX4 tpk3::LEU2

This study

yak1::URA3 TP544n

MATα tpk1::KanMX4 tpk2::KanMX4 tpk3::LEU2

This study

yak1::URA3 TP561a,m

MATa /alpha tpk1::KanMX4/tpk1::KanMX4

This study

tpk2::KanMX4/tpk2::KanMX4 tpk3::LEU2/TPK3 yak1::URA3/YAK1 krh1::HIS3/KRH1 krh2::HIS3/KRH2 TP571o

MATα tpk1::KanMX4 tpk2::KanMX4 tpk3::LEU2

This study

yak1::URA3 krh1::HIS3 krh2::HIS3 TP572o

MATa tpk1::KanMX4 tpk2::KanMX4 tpk3::LEU2

This study

yak1::URA3 krh1::HIS3 krh2::HIS TP573a,p

MATa yak1::URA3 HIS3 LEU2

This study

TP575a

MATα krh1::LEU2 krh2::HIS3 yak1::URA3

This study

TP488p,q

MATa HIS3 LEU2

This study

TP446a

MATa krh1::LEU2 krh2::HIS3

This study

TP448a,m

MATa/α tpk2::KanMX4/TPK2 tpk3::LEU2/TPK3

This study

krh1::LEU2/KRH1 krh2::HIS3/KRH2 TP462r

MATa tpk2::KanMX4 tpk3::LEU2 krh1::LEU2

This study

krh2::HIS3 TP449a,m

MATa/α tpk1::KanMX4/TPK1 tpk3::LEU2/TPK3

This study

krh1::LEU2/KRH1 krh2::HIS3/KRH2 TP461s

MATα tpk1::KanMX4 tpk3::LEU2 krh1::LEU2

This study

krh2::HIS3 TP450a,m

MATa /α tpk1::KanMX4/TPK1 tpk2::KanMX4/TPK2

This study

krh1::LEU2/KRH1 krh2::HIS3/KRH2 TP465t

MATa tpk1::KanMX4 tpk2::KanMX4 krh1::LEU2

This study

krh2::HIS3 TP482a,m,p

MATa tpk2::KanMX4 tpk3::LEU2 HIS3

This study

TP491a,m,p

MATα tpk1::KanMX4 tpk3::LEU2 HIS3

This study

TP486m,p,q

MATa tpk1::KanMX4 tpk2::KanMX4 LEU2 HIS3

This study

MV600u

MATa KRH1(HA)3::URA3

This study

MV601v

MATa HIS3MX6::P(GAL1)(HA)3CYR1

This study

MV602v

MATa HIS3MX6::P(GAL1)GST-GPA2

This study

Y00152

MATa leu2 met15 ura3 his3 gpa2Δ::KanMX4

Ref.5

MV603k

MATa gpa2Δ::KanMX4 GPA2(Q300L)::LEU2

This study

MV604k,v

MATa gpa2Δ::KanMX4 HIS3MX6::P(GAL1)GST-

This study

GPA2(Q300L)::LEU2

MV605u,v

MATa HIS3MX6::P(GAL1)GST-GPA2

This study

KRH1(HA)3::URA3 MV606k,u,v

MATa gpa2Δ::KanMX4 HIS3MX6::P(GAL1)GST-

This study

GPA2(Q300L):: LEU2 KRH1(HA)3::URA3 MV607w

MATa [YEpGFP-HA-BCY1]

This study

MV608u,w

MATa [YEpGFP-HA-BCY1] KRH1-HA3::URA3

This study

pJ69-4A

MATa leu2-3,112 ura3-52 trp1-901 his3-200 gal4Δ

Ref.6

gal80Δ GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7LacZ TP636a

pJ69-4A krh1::URA3

This study

TP637a,b

pJ69-4A krh1::URA3 krh2::KanMX4

This study

TP608a,m,x

MATa/α tpk1::KanMX4/tpk1::KanMX4

This study

tpk2::KanMX4/tpk2::KanMX4 tpk3::LEU2/TPK3 krh1::HIS3/KRH1 krh2::HIS3/KRH2 mouseCα2::URA3 TP665y

MATa tpk1::KanMX4 tpk2::KanMX4 tpk3::LEU2

This study

mouseCα2::URA3 TP667y

MATa tpk1::KanMX4 tpk2::KanMX4 tpk3::LEU2

This study

krh1::HIS3 krh2::HIS3 mouseCα2::URA3 SP1

MATa leu2 his3 trp1 ura3 ade8 can1

Ref.7

MWY63

MATα leu2 his3 trp1 ura3 ade8 tpk1::URA3 tpk2-

Ref.8

63(ts) tpk3::TRP1 bcy1::LEU2 MV620a,b

MWY63 krh1::KanMX4 krh2::HIS3

This study

TP701i

S18-1D RAS2(G19V)::LEU2

This study

a

Plasmids of the pRS series were used to generate PCR disruption cassettes (4).

b

Genomic DNA of Y00351 or Y11668 was used as a template for PCR of a

krh1::KanMX or krh2::KanMX4 disruption cassette, respectively. c

A linear fragment of pUC19-gpa2::LEU2 was used as disruption cassette.

d

Tetrad segregant of a diploid strain generated by crossing Y00351 with Y11668.

e

A linear fragment of ppde2::URA3 was used as disruption cassette (9).

f

Disruptions were made in strain Y26773 (5).

g

Tetrad segregant of strain TP380.

h

Tetrad segregant of strain TP420.

i

YIplac128-RASG19V was cut with PstI for direct integration at the RAS2 promoter

locus. j

Tetrad segregant of strain TP522.

k

YIplac128-GPA2Q300L was cut with MluI for direct integration at the GPA2 promoter

locus. l

Tetrad segregant of strain TP467.

m

tpk1::KanMX4 and/or tpk2::KanMX4 derived from YO1261 and/or Y11089

respectively (5). n

Tetrad segregant of strain TP538.

o

Tetrad segregant of strain TP561.

p

pJJ215 was cut with NsiI for direct integration at the HIS3 terminator locus.

q

pYIplac128 was cut with PflMI for direct integration at the LEU2 promoter locus.

r

Tetrad segregant of strain TP448.

s

Tetrad segregant of strain TP449.

t

Tetrad segregant of strain TP450.

u

pYX012/KRH1 was digested with SphI to direct integration of P(TPI1)-KRH1-HA3

at the TPI1 promoter locus. v

The HIS3MX6GAL1-GST or HIS3MX6GAL1-HA3 cassette (10) was used to replace

the endogenous promoter with GAL1 and an in-frame N-terminal GST fusion. w

Constructed by direct transformation with plasmid YEplac181-GFP-HA-BCY1

(provided by G. Griffioen, KU Leuven). x

YIp-PromTPK1-HA3-mouseCα2-TermTPK1 was cut with AgeI for direct integration at

the TPK1 promoter locus. y

Tetrad segregant of strain TP608.