Title Calcineurin/Crz1 destabilizes Msn2 and Msn4 in the nucleus inresponse to Ca(2+) in Saccharomyces cerevisiae.

Author(s) Takatsume, Yoshifumi; Ohdate, Takumi; Maeta, Kazuhiro;Nomura, Wataru; Izawa, Shingo; Inoue, Yoshiharu

Citation The Biochemical journal (2010), 427(2): 275-287

Issue Date 2010-04-15

URL http://hdl.handle.net/2433/123771

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© The Authors Journal compilation © 2010 BiochemicalSociety; The Version of Record (VoR) is available atwww.biochemj.org; 許諾条件により本文は2010-10-15に公開.; This is not the published version. Please cite only thepublished version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。

Type Journal Article

Textversion author

Kyoto University

WT hog1∆ msn2∆msn4∆

FK50

6 Ca

2+

Ca2+

+ F

K506

Cont

rol

GLO1

ACT1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0 G

lo1

activ

ity (U

/mg

prot

ein)

WT ssk1∆ sho1∆ ssk1∆sho1∆ pbs2∆ hog1∆

Control FK506 Ca2+

Ca2+ + FK506

A

B

FK50

6 Ca

2+

Ca2+

+ F

K506

Cont

rol

FK50

6 Ca

2+

Ca2+

+ F

K506

Cont

rol

Fig. 1

Glo

1 ac

tivity

(U/m

g)

cnb1∆ ACT1

crz1∆

GLO1

0

0.5

1.0

FK506

Na+ Ca2+ Na+ Ca2+ Na+ Ca2+

WT cnb1∆ crz1∆

B

A

FK50

6 Ca

2+

Ca2+

+ F

K506

Cont

rol

FK50

6 Ca

2+

Ca2+

+ F

K506

Cont

rol

Fig. 2

FK506

GLO1

ACT1

w/o CHX w/ CHX

CaCl2

C

Fig. 3

GLO1

ACT1

GLO1

ACT1

GLO1

ACT1

0 5 10 15 20 25 30 40 50 60

WT/Ca2+

WT/Ca2++ FK506

cnb1∆/Ca2+

(min)

0 45 30

Control FK506 Ca2+ Ca2+ + FK506

Hog1-GFP

WT cnb1∆ crz1∆

α-pp38

α-Hog1

Hog1-GFP

B

A

C

Fig. 4

20 10 60 (min)

Ca2+ + FK506

Ca2+

Ca2+

Ca2+

WT

cnb1∆

crz1∆

lacZ PMS1 STRE1 STRE2 AGATG AGGGG

ATG

lacZ PMS2 STRE1 STRE2 AGATG AGGGG

ATG

lacZ PMS12 STRE1 STRE2 AGATG AGATG

ATG

-432 -229

lacZ WT STRE1 STRE2 AGGGG AGGGG

ATG

-428 -225

0

10000

20000

30000

40000

β-G

alac

tosid

ase

(U/m

g)

Fig. 5

0

200

400

600

800

β-G

alac

tosid

ase

(U/m

g)

WT PMS1 PMS2 PMS12 WT PMS1 PMS2 PMS12

WT hog1∆

A

B

Ca2+ Ca2+ + FK506

FK506 Control

Ca2+ Ca2+ + FK506

FK506 Control

C

Ca2+ Ca2+ + FK506

FK506 Control

β-G

alac

tosid

ase

(U/m

g)

WT PMS1 PMS2 PMS12

pRS423

WT PMS1 PMS2 PMS12 0

5000

10000

15000

20000

pRS423+HOG1

Fig. 5

D

0 10

20

30

40

50

60

70

80

90

100

STRECTT1-lacZ

Ca2+ FK506

WT hog1∆

β-G

alac

tosid

ase

(U/m

g)

A C

0

100

200

300

400

500

600

700

GPX1-lacZ

Ca2+ FK506

WT

β-G

alac

tosid

ase

(U/m

g)

Fig. 6

msn2∆msn4∆

0 2

4

6

8

10

12

14

16

18

20

Ca2+ FK506

WT crz1∆

Cata

lase

(U/m

g)

cnb1∆ hog1∆

B

Glo

1 (U

/mg)

0

0.5

1.0

Vector MSN2-OP

A

Ca2+ FK506

Vector CRZ1-OP

Fig. 7

Ca2+

Ca2+ + FK506

0 10 20 30 45 60 Msn2-GFP

(min)

Ca2+

0 10 20 30 45 60 Msn2-GFP

(min)

WT

cnb1∆

B

GFP-Crz1

CaCl2

NaCl +FK506 Control −FK506

C

Ca2+ + FK506

Fig. 8

1 2 3 4 5

Msn2

Cdc28

A

E

Msn2

Cdc28

Msn2

Cdc28

WT

crz1∆

+CaCl2 0 10 30 45 60 90 (min) 0 10 30 45 60 90 w/o FK506 w/ FK506

Msn4

Cdc28

WT +CaCl2 0 10 30 45 60 90 (min) 0 10 30 45 60 90

w/o FK506 w/ FK506

B

Msn2

Cdc28

0 10 30 45 60 90 0 10 30 45 60 90

Msn2

Cdc28

0 10 30 45 60 90 0 10 30 45 60 90

Msn2

Cdc28

0 10 30 45 60 90 0 10 30 45 60 90

Msn2

Cdc28

0 10 30 45 60 90 0 10 30 45 60 90

w/o FK506 w/ FK506 (min)

(min) hog1∆

WT

w/o FK506 w/ FK506

+CaCl2

+CaCl2 +CaCl2 0 10 30 45 60 90 (min) 0 10 30 45 60 90 w/o FK506 w/ FK506

pRS423

pRS423+HOG1

w/o FK506 w/ FK506

w/o FK506 w/ FK506

+CaCl2

+CaCl2

C

D (min)

(min)

STRE

GLO1, CTT1, etc.

Msn2 Hog1 Crz1

Destabilization?

Ypd1 Ssk1

Ssk2/Ssk22

Pbs2

Hog1

Ste11

Sln1 Sho1

Ste20

Ca2+

Ca2+/calmodulin

Calcineurin

Crz1- P

FK506 Msn2

Ca2+ Cytoplasm

Nucleus

Cell membrane

Fig. 9

P

Sln1

bra

nch

Sho1

bra

nch

HOG

MAP

K ca

scad

e