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A disordered protein domain involved in morphogenesis and cell cycle progression.

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(1)

A disordered protein domain involved in morphogenesis and cell cycle

progression.

A. Dagkessamanskaia, K. El Azzouzi, J. François & H.

Martin-Yken.

Laboratoire d’Ingénierie des Systèmes Biologiques et Procédés, UMR- CNRS 5504 & UMR-INRA 792, Toulouse, France.

Email: helene.martin@insa-toulouse.fr Tel: +33 5 61 55 99 59 Web site: http://biopuce.insa-toulouse.fr/jmflab/

Levures Modèles et Outils IX, Strasbourg 2010.

(2)

Introduction

Cell integrity and morphogenesis mechanisms under stress conditions Positionning of Knr4 protein in these mechanisms

Knr4 protein structural information

New Results

Role of N-term in

-protein localization during mitosis and shmoo formation -Morphogenesis / cell cycle control

Connections with the Calcineurin Pathway Effect on the Morphogenesis Checkpoint

Conclusions

(3)

Introduction :

Cell integrity and morphogenesis mechanisms under stress conditions

SBF Rlm1 Pkc1

Slt2 Bck1

MAP kinases cascade

Slt2

Nucleus Mkk1/Mkk2

Sensors

Cell Wall

Plasma Membrane

Calcineurin

Morphogenensis checkpoint,

Cell wall synthesis, Cell cycle progression Calmodulin

Ca

2+

Crz1 Crz1 Phosphatases

Other effectors

Rho1

(4)

(from Sugiura et al., Genes to Cells,7, 619-627, 2007)

Slt2/Mpk1 and calcineurin dependant pathways share

at least one essential function … But wich one ????

(5)

A common function of Slt2/Mpk1 and calcineurin, essential in case of cell wall stress,

the « Morphogenesis checkpoint »

Minuzuma et al., 1998, Nature 392 Corrected by Harrison et al, 2001, Nature Cell Biol, 3, 417-420.

Upon cell wall synthesis or actin polarization defect, this checkpoint maintains cells in G2 until they

adapt and become able

to bud correctly.

(6)

SBF Rlm1

Pkc1

Slt2

Bck1 MAP kinases cascade

Slt2 Nucleus

Mkk1/Mkk2 Knr4

Sensors Cell Wall

Plasma Membrane

Calcineurin Calmodulin Ca

2+

Crz1

Crz1 Cell wall synthesis,

Cell cycle

progression

(7)

3 Identified Domains in Knr4p :

Poorly

structured Structured and globular Unstructured

1 80 340 505

(F. Durand et al. Yeast, 2008)

1 505

These in silico predictions have been confirmed by in vitro experiments.

Collaboration with Pr. Vladimir Uversky

C-H Plot PONDR Analysis

Knr4(340-505) Knr4(1-80) Knr4(80-340) Knr4(1-505) structurées

déstructurées

structurée déstructurée

Knr4p Amino Acids

Score PONDR

80-340 340-505

1-80

In silico Structure Predictions

(8)

Knr4 N-term is required for its localization during vegetative growth

Knr4 without N-term (80-505) Knr4 full (1-505)-GFP

Knr4 without C-term (1-340)-GFP GFP alone

(9)

Knr4 full (1-505)-GFP Knr4 without N-term (80-505)

Knr4 without C-term (1-340)-GFP GFP alone

Knr4 N-term is required for its localization

at the shmoo tip

(10)

Knr4 N-term is required for cell cycle arrest and shmoo formation kinetics

% Unbudded cells % Budding cells % Shmoos

Knr4D-pRS315 5 77 18

Knr4D-pRS315-KNR4 (1-505) 7 17 76

Knr4D-pRS315-KNR4 (1-340) 9 16 75

Knr4D-pRS315-KNR4 (80-505) 4 75 21

(11)

KNR4 and the Calcineurin pathway

- KNR4 and calmodulin were first isolated together by Fishel et al., 1993, and they show similar cellular localization.

- Knr4 and several of its homologs from other species

(Aspergillus fumigatus, Kluveromyces lactis, Cryptococcus

neoformans,…) share a « Calcineurin like phosphoesterase » domain.

- Knr4 D is SL with PKC1/SLT2 pathway members and with

those of the calcineurin signalling pathway, as well as with

drugs that inhibit calcineurin.

(12)

Parsons et al.,2004, Nature Biotech, 22(1) 37-8

A « Chemical-genetic »

screen closely links

KNR4 and Calcineurin

(13)

Knr4 physically interacts with calcineurin

pGAD2f / pOBD2 (negative control) pGAD2f / pOBD2-KNR4(1-505) pGAD2f-CNB1 / pPOBD2

pGAD2f-CNB1 / pPOBD2-KNR4 (1-505) pGAD2f-CNA1 / pOBD2

pGAD2f-CNA1 / pOBD2-KNR4 (1-505) Positive control

b- galactosidase activity (nM of ONPG /min/mg proteins)

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

(14)

Knr4 physically interacts with calcineurin

pGAD2f / pOBD2 (negative control) pGAD2f / pOBD2-KNR4(1-505) pGAD2f-CNB1 / pPOBD2

pGAD2f-CNB1 / pPOBD2-KNR4 (1-505) pGAD2f-CNA1 / pOBD2

pGAD2f-CNA1 / pOBD2-KNR4 (1-505) pGAD2f / pOBD2-KNR4(80-505)

pGAD2f-CNA1 / pOBD2-KNR4 (80-505) Positive control

b- galactosidase activity (nM of ONPG /min/mg proteins)

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

(15)

Knr4 physically interacts with calcineurin

pGAD2f / pOBD2 (negative control) pGAD2f / pOBD2-KNR4(1-505) pGAD2f-CNB1 / pPOBD2

pGAD2f-CNB1 / pPOBD2-KNR4 (1-505) pGAD2f-CNA1 / pOBD2

pGAD2f-CNA1 / pOBD2-KNR4 (1-505) pGAD2f / pOBD2-KNR4(80-505)

pGAD2f-CNA1 / pOBD2-KNR4 (80-505) pGAD2f / pOBD2-KNR4(1-340)

pGAD2f-CNA1 / pOBD2-KNR4 (1-340) Positive control

b- galactosidase activity (nM of ONPG /min/mg proteins)

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

(16)

Knr4 physically interacts with calcineurin

pGAD2f / pOBD2 (negative control) pGAD2f / pOBD2-KNR4(1-505) pGAD2f-CNB1 / pPOBD2

pGAD2f-CNB1 / pPOBD2-KNR4 (1-505) pGAD2f-CNA1 / pOBD2

pGAD2f-CNA1 / pOBD2-KNR4 (1-505) pGAD2f / pOBD2-KNR4(80-505)

pGAD2f-CNA1 / pOBD2-KNR4 (80-505) pGAD2f / pOBD2-KNR4(1-340)

pGAD2f-CNA1 / pOBD2-KNR4 (1-340) pGAD2f / pOBD2-KNR4(1-80)

pGAD2f-CNA1 / pOBD2-KNR4 (1-80) Positive control

b- galactosidase activity (nM of ONPG /min/mg proteins)

0,00 20,00 40,00 60,00 80,00 100,00

13 12 11 10 9 8 7 6 5 4 3 2 1

(17)

A common function of Slt2/Mpk1 and calcineurin, essential in case of cell wall stress,

the « Morphogenesis checkpoint »

Minuzuma et al., 1998, Nature 392 Corrected by Harrison et al, 2001, Nature Cell Biol, 3, 417-420.

Upon cell wall synthesis or actin polarization defect, this checkpoint maintains cells in G2 until they

adapt and become able to bud correctly.

Is this checkpoint functioning in the knr4 D mutant?

(cf : several cell cycle defect phenotypes in this mutant… )

Tested in Tokyo,

in the lab of Pr Yoshikazu Ohya.

(18)

Knr4 participates in the

morphogenesis checkpoint

(19)

Conclusions :

Knr4 links two parallel signalling pathways

SBF Rlm1

Pkc1

Slt2

Bck1 Signalling

cascades

Slt2

Nucleus Mkk1/Mkk2

Knr4

Sensors Cell Wall

Plasma Membrane

Calcineurin Calmodulin

Ca

2+

Crz1

Morphogenensis checkpoint,

Cell wall synthesis, Cell cycle

progression

(20)

Conclusions :

Two disordered domains with very different properties

Essential :

- for protein localization

- when PKC1 pathway is disrupted, - for interaction with calcineurin, - and calcineurin activity .

N-term:

unstructured Structured and globular C-term: unstructured

1 80 505

Interactions inhibition, PEST sequences.

(most of the protein biological function)

340

(21)

Thank you for your attention !

For more data : - Poster session.

- Dagkessamanskaia et al., 2010. Yeast, July 2.

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth,

- Dagkessamanskaia et al., 2010. Protein Science, 19(7):1376-85.

Functional dissection of an intrinsically disordered protein:

understanding the roles of different domains of Knr4 protein in protein-

protein interactions.

(22)

BY4741+ pSG2

BY4741 + pSG2 / CFW crz1D + pSG2

crz1D + pSG2 / CFW knr4D + pSG2

knr4D + pSG2 / CFW

knr4D + pKNR4(1-505) + pSG2

knr4D + pKNR4(1-505) + pSG2 / CFW knr4D + pKNR4(1-340) + pSG2

knr4D + pKNR4(1-340) + pSG2 / CFW knr4D + pKNR4(80-505) + pSG2

knr4D + pKNR4(80-505) + pSG2 / CFW

Specific b- galactosidase activity (nM of ONPG /min/mg proteins)

Crz1 transcriptional activity upon CFW induction is

reduced in the absence of Knr4 protein or its N-terminal domain.

23,99 6,36

0,68 0,70

1,69

9,75 3,78

25,27

38,97 7,51

7,52 4,20

0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00

1 2 3 4 5 6 7 8 9 10 11 12

(23)

Knr4 (1-505)-GFP, a factor 2 hours

(24)

Knr4 (1-505)-GFP, a factor 3 hours

(25)

knr4D mutant + pRS315 Knr4 1-505, a-factor 1h30

(26)

knr4D mutant + pRS315 1h30

(27)

knr4D mutant + pRS315 Knr4 1-340, 1h30

(28)

knr4D mutant + pRS315 Knr4 80-505 1h30

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