L8

Question 1

Functions of cyclin-dependent kinase inhibitors (CKIs)

Answer 1

Cdc28 (S. cerevisiae) and Cdc2 (S. pombe) belong to the group of eukaryotic kinases called cyclin-dependent kinases (CDKs)

Cyclin-dependent kinase inhibitors (CKIs) bind and inactivate the cyclin bound forms of CDKs

Question 2

CKIs in S. cerevisiae

Answer 2

Far1 – binds and inhibits Cdc28-Cln complexes

Sic1 – binds and inhibits Cdc28-Clb complexes

Question 3

CKIs in S. pombe

Answer 3

Rum1 - binds and inhibits Cdc2-Cdc13 (CyclinB) complexes

Question 4

What does Far1 do in S. cerevisiae?

Answer 4

Binds and inhibits Cdc28-Cln complexes

Question 5

What does Sic1 do in S. cerevisiae?

Answer 5

Binds and inhibits Cdc28-Clb complexes

Question 6

What does Rum1 do in S. pombe

Answer 6

Binds and inhibits Cdc2-Cdc13 (CyclinB) complexes

Question 7

Why is it important that the CKI are specific to a cyclin?

Answer 7

It allows you to block specific points in the cell cycle

Question 8

What does a cdc7-ts mutation do in S. cerevisiae?

Answer 8

cdc7-ts mutation blocks right before replication – doesn’t stop budding or spindle pole body duplication

Question 9

What does a cdc4-ts, cdc34-ts or a cdc53-ts mutation do in S. cerevisiae?

Answer 9

Role of cdc4-ts, cdc34-ts, cdc53-ts is analogous

Not involved in replication but isn’t needed for budding or spindle pole body duplication

Still blocks DNA replication

Question 10

Sic1 and the regulation of initiation of DNA replication

Answer 10

CDC34 encodes a ubiquitin conjugating enzyme suggesting protein degradation is important for the initiation of DNA replication

When you knock out the activity of the ability of a protein to add ubiquitin, then you’re not degrading something that you should be degrading before replication occurs

This suggests an inhibitor – needs to be got rid of by ubiquitination before replication occurs

Question 11

Biochemical & genetic evidence that Sic1 is the inhibitor of Cdc28-Clb complexes

Answer 11

In cdc34-ts mutation Cdc28-Cln activity is high. However, mutations which increase stability of G1 cyclins do not arrest cells.

Strains deleted for all six CLB genes looks like cdc34-ts mutations

In the cdc34-ts mutant at the non-permissive temperature no Cdc28-Clb activity can be detected

Sic1 protein is present in early G1, vanishes shortly before S phase and doesn’t reappear until nuclear division.

Sic1 protein is at high levels in cdc34ts mutants at the non-permissive temperature

Question 12

What is the essential function of Cdc28-Cln at START?

Answer 12

To target Sic1 for degradation

cln1Dcln2Dcln3D cells are dead but are rescued by deletion of the SIC1 gene

Will divide & undergo cell division

Question 13

Regulation of Sic1

Answer 13

The transcription of the SIC1 gene is present throughout the cell cycle but peaks at the end of mitosis

Cdc28-Cln phosphorylates Sic1 on multiple sites and this targets it for degradation via Cdc4, Cdc34 and Cdc53
– Sic1 has 9 phosphorylation sites of Cdc28-Cln
– It only gets degraded when any 6 of the 9 are phosphorylated
– Sic1 then disappears & the cell can enter S phase

Question 14

How many phosphorylation sites does Sic1 have on Cdc28-Cln?

Answer 14

9

It only gets degraded when any 6 of the 9 are degraded

Question 15

Function of Sic1

Answer 15

sic1∆ mutants are not dead but enter S phase early and have a longer S phase

Hence Sic1 is involved in the timing of S phase along with the other elements of initiation of DNA replication induced at START

Don’t want to spend lots of time in S phase – lots of opportunity for DNA damage

Question 16

What options does S. cerevisiae have in G1 phase?

Answer 16

Starved cells enter G0 arrest

Haploid cells mate

Diploid cells sporulate

Question 17

What does mating pheromone do?

Answer 17

Mating pheromone inhibits START blocking budding, DNA replication & spindle pole body duplication

Question 18

What happens if we add mating pheromones to yeast cells of 2 different mating types – ‘a’ and alpha?

Answer 18

It interacts with the cell of the opposite type

When it does that it inhibits START – don’t get budding, replication or spindle pole body duplication

It behaves like a cdc28 ts mutation
Behaves like an inhibited triplet Cln mutations

Mating pheromone interferes with the cdc28-G1 cyclins in some way

Question 19

Why do haploid cells arrest the cell division cycle in G1?

Answer 19

If you have a cell in G1 mating with a cell in G2 then you’re making something that has 3 copies of the chromosomes – not making a diploid

If you take a cell in G2 with another G2 cell, then you get 4 copies

By mating 2 cells in G1 you get a diploid

Only phase in the cell cycle when you can mate to get a diploid cell is in G1

Coordinate the cell division cycles with each other

Question 20

How do haploid cells arrest the cell division cycle in G1?

Answer 20

Mating pheromone from one cell in G1 is ejected into the media & arrests the other cell of the opposite mating type

Both act on each other to arrest the cell division cycle – coordinate with each other to allow mating in G1

Question 21

What controls the cell cycle arrest of haploid cells in G1?

Answer 21

The cell cycle arrest depends on a protein kinase cascade and the function of a CKI called Far1 which inhibits Cdc28-Cln1 and Cdc28-Cln2

Question 22

Regulation of Far1

Answer 22

The expression of Far1 is regulated by the Ste12 transcription factor

Far1 is phosphorylated by Fus3 & this possibly activates the Far1 protein

Far1 has several layers of regulation

Question 23

What transcription factor regulates Far1?

Answer 23

Ste1 transcription factor

Question 24

What is Far1 phosphorylated by?

Answer 24

Fus3

This possibly activates the Far1 protein

Question 25

Function of Far1?

Answer 25

Far1 blocks the activity of Cdc28-Cln1 and Cdc28-Cln2 The mechanism of action of Far1 is unclear The phosphorylated form of Far1 appears to bind the Cdc28-Cln1 and Cdc28-Cln2 complexes and induce the degradation of Cln1 and Cln2 Fus3 may phosphorylate Cln3 and target it for degradation This would block Cln1 and Cln2 expression

Question 26

How does the mating pheromone work?

Answer 26

1) 2 mating pheromones – ‘a’ cells look for an alpha cell The alpha factor binds to a receptor on the cell surface of the ‘a’ cell called the Ste2 2) ‘a’ factor binds to a receptor on surface of the alpha cell called Ste3 3) Ensures that an ‘a’ cell & ‘a’ cell don’t mate & an alpha & an alpha don’t mate 4) Can only detect the opposite mating pheromone 5) Then all feeds into a common pathway 6) Once bound to other pheromone it activates the trimeric binding protein releasing the G-alpha bound to GTP 7) Beta-gamma complex binds to a protein kinase called Ste20 which activates a MAP kinase cascade which is called Ste11, Ste7 & Fus3 8) Ste5 is a scaffold pathway for the cascade – holds the proteins together – gives a strong amplification signal

Question 27

What are the 2 outcomes of mating pheromones?

Answer 27

1. Phosphorylates Ste12 – activates the transcription factor and induces expression of Far1 (the inhibitor) 2. Also directly phosphorylates Far1 – this inhibits cdc28-Cln kinase Ensures both cells are in G1

Question 28

Role of Rum1 (a CKI) in S. pombe

Answer 28

Appears to be to prevent mitosis in the absence of DNA replication Rum1 also appears to have a role in size control at START Makes sure mitosis only occurs after replication has occurred

Question 29

Biochemical and genetic evidence of Rum1 function

Answer 29

Overexpression of rum1+ stimulates DNA replication in the absence of mitosis A rum1∆ strain undergoes mitosis if blocked at START Cdc2-Cdc13 is required for the G2/M transition Rum1 can inhibit Cdc2-Cdc13 form of the kinase in vitro A rum1∆ strain has a shorter G1 phase

Question 30

Regulation of Rum1

Answer 30

The Rum1 protein is present only in G1 phase and is absent in S, G2 and M phases Expression levels are relatively constant hence cell cycle regulation is presumably translation and/or proteolytic Protein acts analogously to Sic1 Regulated similar to Sic1

Question 31

CDKs are regulated by a variety of mechanisms, what are they?

Answer 31

* Cyclins * Protein kinases * Protein phosphatases * CKIs * Gene expression * Protein degradation