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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