L3 Flashcards
What is Cdc28?
Serine/threonine protein kinase
What are the G1 cyclins?
Cln1
Cln2
Cln3
Regulators of the Cdc28 substrate specificity
Deletion of the G1 cyclin genes?
Deletion of any one of them individually is not lethal - cells are still viable but don’t grow completely normally
They have overlapping functions
Deletion of all 3 of them at the same time is lethal
Has phenotype same as that of a Cdc28-ts mutation
Cells arrest in G1
mRNA and protein levels for G1 cyclins as you go through the cell cycle
Cln1 and Cln2 expression peaks at the G1 boundary
Cell knows it needs to increase gene expression
As the cell moves into S phase, they turn off
Protein appears and disappears throughout the cycle
Has to have a mechanism for turning the protein over at an extremely high rate meaning it is very unstable
How is Cln1 and Cln2 expression regulated at START?
2 transcription factor complexes: SBF and MBF
How is Cln3 expression regulated at START?
Cln3 is regulated by several mechanisms which include transcriptional control
Some of the Cln3 regulation is linked to nutrient levels and cAMP
Recent studies have shown Hsp70/Hsp40 chaperone/co-chaperone regulation of Cdc28-Cln3 complexes at START
What is SBF made of?
Swi6
Swi4
Regulate G1 cyclin genes
HO gene
What is MBF made of?
Swi6
Mbp1
Regulate G1 cyclin genes Regulates lots of other genes: • Spindle pole body • DNA synthesis • Cell wall components
How are the transcription factors SBF and MBF regulated?
Cdc28 and Cln3 activates both transcription factors
Kinase is regulated by signal transduction ensuring cell size, cAMP and nutrient levels are correct
What is Whi5?
A negative regulator of SBF
When the Cdc28-Cln3 kinase is activated it phosphorylates Whi5
This causes it to come off of the SBF complex and is driven out of the nucleus until its needed to re-establish repression
Regulation of Cdc28-Cln3
Hsp70 = chaperoning
Hsp40 = co-chaperone
One of the Hsp70 proteins in yeast is Ssa1
Cln3 is localised on the ER when deactivated - physically interacting with Ssa1 - Cln3 has a sequence that looks like Hsp40
Ssa1 conned into thinking its binding a Hsp40 protein
Ydj1 is the normal binding partner for Ssa1
As levels of Ydj1 start to increase, it binds Ssa1 a lot better and knocks off the complex
Once released it can go into the nucleus where it can activate transcription
What happens if you overexposes Ydj1?
It will drive cells through G1 faster
Drives the complex off of Ssa1 a lot faster
Proportion of cells in G1 drops
Regulation of START
Cell has to be the right size and has to decided whether o not it wants to enter the CC
Mainly decided by the activity of the cyclin-dependent kinases and the G1 cyclins
Cell size, nutrients and cAMP all regulate the activity of `cdc28-Cln3
They drive the CC by activating SBF and MBF transcription factors which then activate the transcription of Cln1 and Cln2
Pheromone response has its own pathway that inhibits the Cdc28-Cln1/2 cyclin
