protein and nucleic acid degradation a set of enzymes targets a protein for degradation by tagging...

Synthesis(transcription and translation)

On OffPhosphorylation Dephosphorylation

Destruction

Methods of cellular regulation

Protein and Nucleic Acid Degradation

A set of enzymes targets a protein for degradation by tagging it with an ~70 amino acid protein called ubiquitin. The ubiquitin tag is recognized by a large protein machine called a proteasome. Upon entering the proteasome, the protein is unfolded and hydrolysed (degraded). Ubiquitin molecules are highly resistant to the proteasome and can be recycled.

E1-Ub E1

E2-Ub E2

E3

Protein

26S Proteosome

ADP

ATPPeptides

UbATP

Ubiquitinated Protein

UbUb

UbUb

UbAMP

(Cyclosome, cdc53 etc.)

Ubiquitin-dependent proteolysis pathway

Deubiquitinating enzymes

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Skp1-Cdc53-F box (+Rbx+cdc34(E2))

Facts:

1. Cln1,2,3 bind Cdk1 in G1 and form an active kinase.

Cells without cln1,2,3 are inviable

2. Clb 5,6 bind Cdk1 in G1 but as long as they are bound

by Sic1 they don’t form an active kinase.

3. Clb5,6-cdk1 activity is essential for DNA replication

Experiment Sic1 Cell cycle phase

Cln- / cdc34- Unphosphorylated

Stable

G1 arrest

Cln+ / cdc34- Phosphorylated

Stable

G1 arrest

Cln- / cdc34+ Unphosphorylated

Stable

G1 arrest

Cln+ / cdc34- Phosphorylated

Degraded

Cycling

Cdc34 acts downstream of Cln, why then does Cln+ / cdc34- andCln- / cdc34+ have the same phenotype ?

Suggest 2 possibilities how cln and cdc34 interact. Which is more likely ?

Anaphase Promoting Complex/Cyclosome

Somatic cell cycle Embryonal cell cycle

protein levels

phasecheckpoint

M S

START

S

START

M

Levels of Cyclin B oscillate during the cell cycle

0 1 10 12 14 16 205 8

Cyclin B1

Cyclin B2

S G2 M G1 S G2 M

STARTSTART MA

65' 70' 75' 80' 85' 90' 95 100

M A M AM A

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Fizzy and Fizzy related family tree

S.pombe slp1S.cerevisiaecdc20

Carrot fzy Drosophilafzy

Ratp55cdc

Humanp55cdc

S.cerevisiaecdh1

Xenopusfzr

Drosophilafzr

Fzr activation of the cyclosome is inhibited bycdk1-cyclin B kinase activity

125I-Cyclin B (Ub)n

125I-Cyclin B

CyclosomeFzr

Cdk1-cyclin B1Staurosporine - - - - - +

1 2 3 4 5 6

- + - + + +- - + + + +- - - - + +

Cdk1-cyclin B phosphorylation of fzrprevents fzr from activating the cyclosome

1 2 3 4

125I-Cyclin B

Incubation II fzrCS

-+

+-

-+

+-

Incubation Ifzr

CScdk -

+-

-

-+

+

+-

+

-+

125I-Cyclin B (Ub) n

APC

Checkpoint

cdk1Cyclin B

Fzy

CheckpointCheckpoint

Fzr

Polo

Prophase

Emi

APC

cdk1Cyclin B

Fzy

CheckpointCheckpoint

Checkpoint

Fzr

Polo

Metaphase

APC

cdk1Cyclin B

Fzr

FzyFzr

Fzy

Anaphase

Polo

cdc14

Metaphase

APC

cdk2Cyclin A

Fzr

G1

Emi

Fzr

S

APC

Checkpoint

cdk1Cyclin B

Fzy

CheckpointCheckpoint

Fzr

Polo

Prophase

Emi

APC

cdk1Cyclin B

Fzy

CheckpointCheckpoint

Checkpoint

Fzr

Polo

Metaphase

APC

cdk1Cyclin B

Fzr

FzyFzr

Fzy

Anaphase

Polo

cdc14

Metaphase

Substrates of the APC/C

• Cyclin A Prometaphase• Cyclin B (1,2,3) Metaphase-anaphase-G1• Securin (Pds1, Cut2) Metaphase-anaphase -G1

Xkid Metaphase-anaphase -G1• AseI Telophase (?)• Cdc6 (metazoan) G1• Geminin ?-G1• Fzy (cdc20) Telophase-G1• Polo (plk1, cdc5)• Ubch10 Telophase

0

20

40

60

80

Hours after release from nocodazole0 7 11 133 5 15 189

100

KAA-DM

KAA WTDM

Securin degradation is mediated both by a d-boxand by a KEN-box

0 3 5 7 9 11 13 15 18

M G1 G2S

Hours after release from nocodazole

securin-KAACAT

securin-KAADM-CAT

securin-DMCAT

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Metaphase

Anaphase

Telophase

Cytokinesis